Delta3, fthma-070, tango85, tango77, spoil, neokine, tango129, and integrin alpha subunit protein and nucleic acid molecules and uses thereof

ABSTRACT

The invention provides novel Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 and A259 polypeptides, proteins, and nucleic acid molecules. In addition to isolated, full-length Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 and A259 proteins, the invention further provides isolated Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 and A259 fusion proteins, antigenic peptides and anti-Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 and A259 antibodies. The invention also provides Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 and A259 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/175,714, filed Jul. 5, 2005, pending.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is acontinuation-in-part of U.S. patent application Ser. No. 10/417,719,filed Apr. 17, 2003, now abandoned, which is a continuation of U.S.patent application Ser. No. 09/568,218, filed May 9, 2000, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 08/872,855, filed Jun. 11, 1997, now U.S. Pat. No. 6,121,045,which is a continuation-in-part of U.S. patent application Ser. No.08/832,633, filed Apr. 4, 1997, now abandoned.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is alsoa continuation-in-part of U.S. patent application Ser. No. 10/895,676,filed Jul. 21, 2004, now abandoned, which is a continuation of U.S.patent application Ser. No. 10/105,934, filed Mar. 25, 2002, nowabandoned, which is a continuation of U.S. patent application Ser. No.09/862,972, filed May 22, 2001, now abandoned, which is a continuationof U.S. patent application Ser. No. 09/062,389, filed Apr. 17, 1998, nowabandoned, which claims the benefit of Provisional Application Ser. No.60/062,017, filed Oct. 10, 1997, now abandoned, and ProvisionalApplication Ser. No. 60/044,746, filed Apr. 18, 1997, now abandoned.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is alsoa continuation-in-part of U.S. patent application Ser. No. 10/095,407,filed Mar. 11, 2002, now abandoned, which is a continuation of U.S.patent application Ser. No. 09/451,828, filed Nov. 30, 1999, nowabandoned, which is a divisional of U.S. patent application Ser. No.09/128,155, filed Aug. 3, 1998, now U.S. Pat. No. 6,117,654, whichclaims the benefit of Provisional Application Ser. No. 60/091,650, filedJul. 2, 1998, now abandoned, and Provisional Application Ser. No.60/054,646, filed Aug. 4, 1997, now abandoned.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is alsoa continuation-in-part of U.S. patent application Ser. No. 10/126,560,filed Apr. 19, 2002, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 09/237,571, filed Jan. 26, 1999, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 09/013,810, filed Jan. 27, 1998, now U.S. Pat. No. 6,197,551.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is alsoa continuation-in-part of U.S. patent application Ser. No. 10/413,899,filed Apr. 14, 2003, now abandoned, which is a divisional of U.S. patentapplication Ser. No. 09/940,240, filed Aug. 27, 2001, now abandoned,which is a continuation of U.S. patent application Ser. No. 09/248,239,filed Feb. 10, 1999, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 09/023,664, filed Feb. 10, 1998, nowabandoned.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is alsoa continuation-in-part of U.S. patent application Ser. No. 10/105,150,filed Mar. 25, 2002, now abandoned, which is a continuation of U.S.patent application Ser. No. 10/060,680, filed Jan. 30, 2002, nowabandoned, which is a continuation of U.S. patent application Ser. No.09/057,951, filed Jan. 8, 2001, now abandoned.

U.S. patent application Ser. No. 11/175,714, filed Jul. 5, 2005, is alsoa continuation-in-part of U.S. patent application Ser. No. 10/601,368,filed Jun. 23, 2003, now abandoned, which is a continuation of U.S.patent application Ser. No. 09/572,003, filed May 15, 2000, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 09/561,263, filed Apr. 27, 2000, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 09/322,790,filed May 28, 1999, now abandoned.

The entire contents of each of the above-listed patent applications areincorporated herein by reference.

The contents of the Sequence Listing are submitted herewith on compactdisc in duplicate. Each duplicate disc has a copy of the file “sequencelisting.txt” which is incorporated herein by this reference. This fileis 961 kilobytes and was copied onto compact disc on Oct. 12, 2007.

BACKGROUND OF THE INVENTION

There is considerable medical interest in secreted andmembrane-associated mammalian proteins. Many such proteins, for example,cytokines, are important for inducing the growth or differentiation ofcells with which they interact or for triggering one or more specificcellular responses.

The demonstrated clinical utility of several secreted proteins in thetreatment of human disease, for example, erythropoietin,granulocyte-macrophage colony stimulating factor (GM-CSF), human growthhormone, and various interleukins, illustrates the importance ofsecreted proteins.

Many membrane-associated proteins are receptors which bind a ligand(s)and transmit an intracellular signal. As such, membrane-associatedproteins can be used to identify (or design) small molecules which actas agonists or antagonists of the ligand.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofcDNA molecules which encode the TANGO 24 (DELTA3), FTHMA-070, TANGO 85,TANGO 77, TANGO 80 (SPOIL), TANGO 112 (NEOKINE), TANGO 129 and A259(INTEGRIN ALPHA SUBUNIT) proteins.

Delta 3

The invention is based at least in part on the discovery of a human geneencoding a novel Delta protein, and its mouse homolog, each of whichdiffers substantially from the previously described Delta proteins.Accordingly, the novel Delta proteins of the invention are referred toherein as Delta3 proteins. Thus, the invention provides Delta3 proteins,and nucleic acids encoding Delta3 proteins. An exemplary human Delta3(hDelta3) is contained in a plasmid which was deposited with the ATCC®on Mar. 5, 1997, and has been assigned ATCC® accession number 98348.

Based on Northern blot analysis of RNA prepared from a number of humantissues, a 3.5 kb message was expressed in fetal brain, lung, liver andkidney; and adult heart, placenta, lung, skeletal muscle, kidney,pancreas, spleen, thymus, prostate, testis, ovary, small intestine andcolon. In addition, the hDelta3 gene was found to be expressed atrelatively high levels in at least some tumor cells (e.g., coloncarcinoma) and its expression can be up-regulated in response to variousgrowth factors (e.g., bFGF and VEGF). Furthermore, the expression ofhDelta3 was also shown to increase in response to a signal-induceddifferentiation of endothelial cells, indicating a role for hDelta3 inmodulating the growth and/or differentiation of cells, in particularendothelial cells.

In situ hybridization of a panel of adult and embryonic tissues using aprobe complementary to mRNA of mDelta3 showed that mDelta3 expressionwas most abundant and widespread during embryogenesis. Strongestexpression was observed in the eye during all stages of embryogenesistested, mDelta3 was also seen in the developing lung, thymus and brownfat. In addition to the focal expression seen above, a multi-focal,scattered pattern was seen throughout the embryo. This signal patternwas more focused in the cortical region of the kidney and outlining theintestinal tract. Adult expression was highest in the ovary and thecortical regions of the kidney and adrenal gland. The expression seenduring embryogenesis indicates that Delta3 has a role in cell growthand/or differentiation.

As demonstrated herein, Delta3 encodes a Notch ligand. In particular,data presented herein demonstrates that hDelta3 encodes a Notch ligand,as it has been shown to block the differentiation of C2C12 into myotubesin a similar fashion to other Notch ligands (e.g., Jagged 1).

As described herein, the hDelta3 gene has been localized by Southernblotting a membrane containing DNA from a panel of a human/hamstermono-chromosomal somatic cell hybrids. The results demonstrate thathuman Delta3 is located on human chromosome 15, close to frameworkmarkers D15S1244 and D15S144, a chromosomal region that has beenassociated with the neurological disease Agenesis of the Corpus Callosumwith Peripheral Neuropathy (ACCPN) (Casaubon et al. (1996) Am J. Hum.Genet. 58:28). Accordingly, polymorphisms in Delta3 are thought to beinvolved in this neurological disease. As described further herein,Delta3 is also likely to be involved in other neurological diseases aswell as in non-neurological diseases.

In one aspect, the invention features isolated Delta3 nucleic acidmolecules, e.g., mammalian, such as human or mouse, Delta3 nucleicacids. The disclosed molecules can be non-coding, (e.g., probe,antisense or ribozyme molecules) or can encode a functional Delta3polypeptide, e.g., a polypeptide which can modulate at least oneactivity of a Delta3 polypeptide. In one embodiment, the nucleic acidmolecules hybridize to the Delta3 gene contained in the plasmid havingAmerican Type Culture Collection (ATCC®) Accession Number 98348. Inanother embodiment, the claimed nucleic acid is capable of hybridizingunder stringent conditions to the nucleotide sequence set forth in SEQID NOS: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or thenucleotide sequence of the cDNA of a clone deposited with the ATCC® asAccession Number 98348, or to the complement thereof.

In further embodiments, the nucleic acid molecule is a Delta3 nucleicacid molecule that is at least about 50%, 55%, 60%, 70%, preferably 80%,more preferably 85%, and even more preferably at least about 95% or 98%identical to the nucleic acids shown in SEQ ID Nos: 1, 3, 24, 26, 27,29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence of thecDNA of a clone deposited with the ATCC® as Accession Number 98348, or acomplement thereof.

In another embodiment, the nucleic acid molecule is a Delta3 nucleicacid that is at least about 50%, 55%, 60%, 65%, 70%, preferably 80%,more preferably 85%, and even more preferably at least about 95% or 98%identical to the nucleic acids shown in SEQ ID NOS: 1, 3, 24, 26, 27,29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence of thecDNA of a clone deposited with the ATCC® as Accession Number 98348 or acomplement thereof, wherein such nucleic acid molecules encodepolypeptides or proteins that exhibit at least one structural and/orfunctional feature of a polypeptide of the invention.

In another embodiment, the nucleic acid molecule encodes a polypeptidethat is at least about 55%, 60%, 70%, preferably 80%, more preferably85%, and even more preferably at least about 90, 95% or 98% identical tothe polypeptide shown in SEQ ID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348, or a complementthereof.

In another embodiment, the nucleic acid molecule encodes a polypeptidethat is at least about 55%, 60%, 70%, preferably 80%, more preferably85%, and even more preferably at least about 90, 95% or 98% identical tothe polypeptide shown in SEQ ID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348, or a complementthereof, wherein such nucleic acid molecules encode polypeptides orproteins that exhibit at least one structural and/or functional featureof a polypeptide of the invention. In another embodiment, the nucleicacid molecule encodes a polypeptide that is at least about 72%,preferably 80%, more preferably 85%, and even more preferably at leastabout 90 or 95% similar to the polypeptide shown in SEQ ID NOS: 2, 25,28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the hDelta 3 cDNA sequencecontained in the plasmid having ATCC® Accession Number 98348, or acomplement thereof.

In another embodiment, the nucleic acid molecule encodes a polypeptidethat is at least about 72%, preferably 80%, more preferably 85%, andeven more preferably at least about 90 or 95% similar to the polypeptideshown in SEQ ID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, orthe Delta 3 cDNA sequence contained in the plasmid having ATCC®Accession Number 98348, or a complement thereof, wherein such nucleicacid molecules encode polypeptides or proteins that exhibit at least onestructural and/or functional feature of a polypeptide of the invention.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of any of SEQID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, the fragmentincluding at least 15 (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 440, 460, 475, 500, 525, 550,575, 600, 625, 650, 675, or 685) contiguous amino acids of any of SEQ IDNOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the polypeptideencoded by the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of any of SEQID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, the fragmentincluding at least 15 (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 440, 460, 475, 500, 525, 550,575, 600, 625, 650, 675, or 685) contiguous amino acids of any of SEQ IDNOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the polypeptideencoded by the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348, wherein the fragment exhibits at least one structuraland/or functional feature of a polypeptide of the invention.

The nucleic acids of the invention can comprise a nucleotide sequenceencoding at least one domain or motif of a Delta3 protein, i.e., adomain or motif selected from the group consisting of an amino-terminalsignal peptide, a Delta-Serrated lag-2 (DSL) domain, Epidermal GrowthFactor (EGF)-like domain 1, EGF-like domain 2, EGF-like domain 3,EGF-like domain 4, EGF-like domain 5, EGF-like domain 6, EGF-like domain7, EGF-like domain 8, a Delta3 extracellular domain, a transmembranedomain (TM), and a cytoplasmic domain. Other nucleic acids of theinvention encode soluble Delta3 proteins, e.g., Delta3 proteinscomprising at least a portion, such as one or more domains, of theextracellular domain of a Delta3 protein. A soluble Delta3 protein is aprotein that is soluble at standard physiological conditions, andincludes, but is not limited to a Delta3 protein that does not comprisea transmembrane domain, e.g., an extracellular Delta3 domain. Thesesoluble polypeptides may or may not comprise a signal peptide. Othersuch nucleic acids encode soluble fusion proteins comprising Delta3amino acid sequence and a heterologous amino acid sequence, e.g., animmunoglobulin constant region.

The invention also provides probes and primers comprising substantiallypurified oligonucleotides, which correspond to a region of nucleotidesequence which hybridizes to at least about 6 consecutive nucleotides ofthe sequences set forth as SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43 or 45, or the nucleotide sequence of the cDNA of a clonedeposited with the ATCC® as Accession Number 98348, or a complementthereof, or naturally-occurring mutants thereof. In preferredembodiments, the probe/primer further includes a label group attachedthereto, which is capable of being detected.

The invention features nucleic acid molecules of at least 425, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, or 2800 nucleotides of the nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of the human Delta3 cDNA clone of ATCC®Accession NO: 98348, or a complement thereof. The invention alsofeatures nucleic acid molecules comprising at least 25, 50, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050 or 2061nucleotides of nucleic acids 1 to 2062 of SEQ ID NO:1, or a complementthereof, wherein such nucleic acid molecules encode polypeptides orproteins that exhibit at least one structural and/or functional featureof a polypeptide of the invention.

The invention features nucleic acid molecules which include a fragmentof at least 340, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1450,1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050or 2058 nucleotides of the nucleotide sequence of SEQ ID NO:3, or acomplement thereof. The invention also features nucleic acid moleculescomprising at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or 1724nucleotides of nucleic acids 1 to 1725 of SEQ ID NO:3, or a complementthereof.

The invention features nucleic acid molecules of at least 480, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1450, 1500, 1550, 1600, 1650, 1700, 1750,1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2300, 2400, 2500, 2600,2700, 2800, 2900, 3000, 3100 or 3130 nucleotides of the nucleotidesequence of SEQ ID NO:24, the nucleotide sequence of the mouse Delta3cDNA, or a complement thereof. The invention also features nucleic acidmolecules comprising at least 25, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, or 1529 nucleotides ofnucleic acids 1 to 1530 of SEQ ID NO:24, or a complement thereof,wherein such nucleic acid molecules encode polypeptides or proteins thatexhibit at least one structural and/or functional feature of apolypeptide of the invention.

The invention features nucleic acid molecules which include a fragmentof at least 415, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100nucleotides of the nucleotide sequence of SEQ ID NO: 26, or a complementthereof. The invention also features nucleic acid molecules comprisingat least 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500 or 1529 nucleotides of nucleic acids 1 to 1530 ofSEQ ID NO: 3, or a complement thereof.

Another aspect of the invention provides vectors, e.g., recombinantexpression vectors, comprising a nucleic acid molecule of the invention.In another embodiment the invention provides host cells containing sucha vector. The invention also provides methods for producing apolypeptide of the invention by culturing, in a suitable medium, a hostcell of the invention containing a recombinant expression vectorencoding a polypeptide of the invention such that the polypeptide of theinvention is produced.

For expression, the subject Delta3 nucleic acids can include a mammaliantranscriptional regulatory sequence, e.g., at least one of atranscriptional promoter (e.g., for constitutive expression or inducibleexpression) or transcriptional enhancer sequence, the regulatorysequence is operably linked to the Delta3 gene sequence. Such regulatorysequences in conjunction with Delta3 nucleic acid molecules can beuseful in vectors for gene expression. This invention also describeshost cells transfected with said expression vectors whether prokaryoticor eukaryotic, also in vitro (e.g., cell culture) and in vivo (e.g.,transgenic) methods for producing Delta3 proteins by employing saidexpression vectors. In a preferred embodiment, the Delta3 nucleic acidsare cloned into a mammalian expression vector, and transfected intomammalian cells. The use of mammalian cells increases the likelihood ofproper protein folding and post-translational modification of theexpressed proteins.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of any of SEQ ID NOS: 1, 3, 24, 26, 27, 29, 31, 33,35, 37, 39, 41, 43 or 45 of the cDNA of a clone deposited with the ATCC®as Accession Number 98348, or a complement thereof, wherein preferablysuch nucleic acid molecules encode polypeptides or proteins that exhibitat least one structural and/or functional feature of a polypeptide ofthe invention. In other embodiments, the nucleic acid molecules are atleast 485 (500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, or 2800) nucleotides in length and hybridize under stringentconditions to a nucleic acid molecule comprising the nucleotide sequenceof any of SEQ ID NOS: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43or 45 of the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348, or a complement thereof.

In preferred embodiments, the isolated nucleic acid molecules encode acytoplasmic, transmembrane, or extracellular domain of a polypeptide ofthe invention.

The invention includes nucleic acid molecules which encode an allelicvariant of a polypeptide comprising the amino acid sequence of any ofSEQ ID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an aminoacid sequence encoded by the cDNA of a clone deposited with the ATCC® asAccession Number 98348, or a complement thereof, wherein the nucleicacid molecule hybridizes under stringent conditions to a nucleic acidmolecule having a nucleic acid sequence encoding any of SEQ ID NOS:1, 3,24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotidesequence of the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348, or a complement thereof.

The invention includes nucleic acid molecules which encode an allelicvariant of a polypeptide comprising the amino acid sequence of any ofSEQ ID NOS: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an aminoacid sequence encoded by the cDNA of a clone deposited with the ATCC® asAccession Number 98348, or a complement thereof, wherein the nucleicacid molecule hybridizes under stringent conditions to a nucleic acidmolecule having a nucleic acid sequence encoding a polypeptide havingany of the amino acid sequences shown in SEQ ID NOS: 2, 25, 28, 30, 32,34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded by thecDNA of a clone deposited with the ATCC® as Accession Number 98348, or acomplement thereof, wherein such nucleic acid molecules encodepolypeptides or proteins that exhibit at least one structural and/orfunctional feature of a polypeptide of the invention.

In one embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 455 is a guanine (G) (SEQ ID NO: 1). In thisembodiment, the amino acid at position 40 is glutamate (E) (SEQ ID NO:2). In another embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 455 is a cytosine (C) (SEQ ID NO: 27). In thisembodiment, the amino acid at position 40 is glutamine (Q) (SEQ ID NO:28). In another embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 455 is a thymidine (T) (SEQ ID NO: 29). In thisembodiment, the amino acid at position 40 is a stop codon (SEQ ID NO:30). In another embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 455 is a adenine (A) (SEQ ID NO: 31). In thisembodiment, the amino acid at position 40 is lysine (K) (SEQ ID NO: 32).

In another embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a nucleic acid ofthe invention.

In another aspect, the invention features isolated Delta3 polypeptides,preferably substantially pure preparations, e.g., of plasma-purified orrecombinantly produced Delta3 polypeptides. Preferred proteins andpolypeptides possess at least one biological activity of thecorresponding naturally-occurring human polypeptide. Such an activitycan be a direct activity, such as an association with or an enzymaticactivity on a second protein or an indirect activity, such as a cellularsignaling activity mediated by interaction of the protein with a secondprotein. Thus, such activities include, for example, ones related toDelta3's function as a Notch ligand. Delta3 activities include, e.g.,(1) the ability to form protein-protein interactions with proteins inthe signaling pathway of the naturally-occurring polypeptide; (2) theability to bind a ligand of the naturally-occurring polypeptide; (3) theability to bind to an intracellular target of the naturally-occurringpolypeptide; (4) the ability to modulate cellular proliferation; (5) theability to modulate cellular differentiation; (6) the ability tomodulate chemotaxis and/or migration; and/or (7) the ability to modulatecell death; (8) maintenance of energy homeostasis (e.g., regulating thebalance or imbalance between energy storage and energy expenditure, forexample, increasing or decreasing energy expenditure; (9) regulation ofadaptive thermogenesis (e.g., regulation of the biogenesis ofmitochondria, regulation of the expression of mitochondrial enzymes,regulation of expression of uncoupling proteins; (10) regulation ofadiposity; (11) modulation of the efficiency of energy storage; (12)regulation of appetite; (13) expansion/reduction of fat mass; (14)regulation of vasculogenesis (blood vessel formation); (15) regulationof tumor angiogenesis; (16) regulation of wound healing; (17)expansion/reduction of tumor mass; (18) the ability to modulatedevelopment, differentiation, proliferation and/or activity of immunecells (e.g., leukocytes and macrophages), endothelial cells and smoothmuscle cells; (19) the ability to modulate the host immune response;(20) the ability to modulate the development of organs, tissues and/orcells of the embryo and/or fetus; (21) the ability to modulate cell-cellinteractions and/or cell-extracellular matrix interactions; (22) theability to modulate atherosclerosis, e.g., the initiation andprogression of atherosclerosis; (23) the ability to modulateatherogenesis; (24) the ability to modulate inflammatory functions e.g.,by modulating leukocyte adhesion to extracellular matrix and/orendothelial cells; (25) the ability to form, e.g., stabilize, promote,facilitate, inhibit, or disrupt, cell to cell and cell to blood productinteraction, e.g., between leukocytes and platelets or leukocytes andvascular endothelial cells; (26) ability to modulate development,differentiation and activity of skeletal muscle cells and tissue; and(27) ability to act in stem cell preservation.

In certain embodiments, the subject polypeptides are capable ofmodulating an activity of a Delta3 polypeptide, e.g., cell growth and/ordifferentiation or induction of apoptosis. In other embodiments, thesubject Delta3 polypeptides can modulate neurogenesis (e.g., byinhibiting Notch expressing cells from becoming committed to a neuralfate). In addition, Delta3 polypeptides which specifically antagonizethe activity of a native Delta3 polypeptide, such as may be provided bytruncation mutants or other dominant negative mutants, are alsospecifically provided herein.

In one embodiment, a polypeptide of the invention has an amino acidsequence sufficiently identical to an identified domain of a polypeptideof the invention. As used herein, the term “sufficiently identical”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., with asimilar side chain) amino acid residues or nucleotides to a second aminoacid or nucleotide sequence such that the first and second amino acid ornucleotide sequences have a common structural domain and/or commonfunctional activity. For example, amino acid or nucleotide sequenceswhich contain a common structural domain having about 65% identity,preferably 75% identity, more preferably 85%, 95%, or 98% identity.

In one embodiment, the polypeptide is identical to a Delta3 proteinrepresented in SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or46, or the amino acid sequence encoded by the cDNA of a clone depositedwith the ATCC® as Accession Number 98348. Preferably, a Delta3polypeptide has an amino acid sequence, which is at least about 55%,60%, 70%, preferably at least about 80%, more preferably at least about90%, and even more preferably at least about 95% or 98% identical to thepolypeptide represented by SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38,40, 42, 44 or 46, or the amino acid sequence encoded by the cDNA of aclone deposited with the ATCC® as Accession Number 98348.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 55%, preferably 65%, 75%,85%, 95%, or 98% identical to the amino acid sequence of any of SEQ IDNOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acidsequence encoded by the cDNA of a clone deposited with the ATCC® asAccession Number 98348, wherein the polypeptides or proteins alsoexhibit at least one structural and/or functional feature of apolypeptide of the invention.

Also within the invention are isolated polypeptides or proteins whichpreferably are encoded by a nucleic acid molecule having a nucleotidesequence that is at least about 55%, more preferably 60%, 65%, 75%, 85%,or 95% identical the nucleic acid sequence encoding any of SEQ ID NOs:2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, wherein thepolypeptides or proteins preferably also exhibit at least one structuraland/or functional feature of a polypeptide of the invention, andisolated polypeptides or proteins which are encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having the sequenceof any of SEQ ID NOs:1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or45, or a complement thereof, or the non-coding strand of the cDNA of aclone deposited with the ATCC® as Accession Number 98348.

In a preferred embodiment, the Delta3 polypeptide is encoded by anucleic acid which hybridizes in high stringency conditions with anucleic acid sequence represented in one of SEQ ID NOs: 1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45 or with the nucleic acidcontained in the plasmid having ATCC® Accession NO: 98348.

The subject Delta3 proteins also include modified proteins, which areresistant to post-translational modification, as for example, due tomutations which alter modification sites (such as tyrosine, threonine,serine or asparagine residues), or which prevent glycosylation of theprotein, or which prevent interaction of the protein with intracellularproteins involved in signal transduction.

The Delta3 polypeptide can comprise a full-length protein, such asrepresented in SEQ ID NO: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or46, or it can comprise a fragment corresponding to one or moreparticular motifs/domains (e.g., an extracellular domain, a DSL domainor an EGF-like domain, all of which are described below), or to othersizes, e.g., at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600 or 650 amino acids in length.

The polypeptides of the invention can comprise at least one domain ormotif of a Delta3 protein, i.e., a domain or motif selected from thegroup consisting of an amino-terminal signal peptide, a Delta-Serratedlag-2 (DSL) domain, Epidermal Growth Factor (EGF)-like domain 1,EGF-like domain 2, EGF-like domain 3, EGF-like domain 4, EGF-like domain5, EGF-like domain 6, EGF-like domain 7, EGF-like domain 8, a Delta3extracellular domain, a transmembrane domain (TM), and a cytoplasmicdomain. Other polypeptides comprise soluble Delta3 proteins, e.g.,Delta3 proteins comprising at least a portion, such as one or moredomains, of the extracellular domain of a Delta3 protein. A solubleDelta3 protein is a protein that is soluble at physiological conditions,and includes but is not limited to a Delta3 protein that does notcomprise a transmembrane domain, e.g., an extracellular Delta3 domain.These soluble polypeptides may or may not comprise a signal peptide.Other such polypeptides comprise soluble fusion proteins comprisingDelta3 amino acid sequence and a heterologous amino acid sequence, e.g.,an immunoglobulin constant region.

In one embodiment, the isolated polypeptide of the invention lacks botha transmembrane and a cytoplasmic domain. In another embodiment, thepolypeptide lacks both a transmembrane domain and a cytoplasmic domainand is soluble under physiological conditions.

Also within the invention are polypeptides which are allelic variants ofa polypeptide that includes the amino acid sequence of any of SEQ IDNOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acidsequence encoded by the cDNA of a clone deposited with the ATCC® asAccession Number 98348, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes under stringent conditions to a nucleicacid molecule having the sequence of any of SEQ ID NOs:1, 3, 24, 26, 27,29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence of thecDNA of a clone deposited with the ATCC® as Accession Number 98348, or acomplement thereof.

Also within the invention are polypeptides which are allelic variants ofa polypeptide that includes the amino acid sequence of any of SEQ IDNOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acidsequence encoded by the cDNA of a clone deposited with the ATCC® asAccession Number 98348, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes under stringent conditions to a nucleicacid molecule having the sequence of any of SEQ ID NOs: 1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence ofthe cDNA of a clone deposited with the ATCC® as Accession Number 98348,or a complement thereof, wherein such nucleic acid molecules encodepolypeptides or proteins that exhibit at least one structural and/orfunctional feature of a polypeptide of the invention.

Another aspect of the invention features fusion proteins comprising aDelta3 amino acid sequence. For instance, the Delta3 protein can beprovided as a recombinant fusion protein which includes a secondpolypeptide portion, e.g., a second polypeptide having an amino acidsequence unrelated (heterologous) to the Delta3 polypeptide, (e.g., thesecond polypeptide portion is glutathione-S-transferase, an enzymaticsequence such as alkaline phosphatase or an epitope tag).

Fusion proteins of the invention include, for example, Delta3immunoglobulin (Delta3-Ig) fusion proteins. For example, a Delta3 fusionprotein can comprise the extracellular portion of a Delta3 protein fusedto the constant region of an immunoglobulin molecule. Preferredextracellular portions comprise at least one domain selected from thegroup consisting of a signal peptide, a DSL domain, and the eightEGF-like domains of a Delta3 protein. An even more preferredextracellular domain comprises an amino acid sequence from amino acid 1to amino acid 529 of SEQ ID NO: 2 or from amino acid 1 to 530 of SEQ IDNO: 25. Yet other preferred Delta3 fusion proteins comprise a portion ofa Delta3 protein that is sufficient for binding to a second protein,such as the DSL domain to a second protein which is, for example, aNotch protein.

Yet another aspect of the present invention concerns an immunogencomprising a Delta3 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for aDelta3 polypeptide, e.g., a humoral response, an antibody responseand/or cellular response. In preferred embodiments, the immunogencomprises an antigenic determinant, e.g., a unique determinant, from theprotein represented by SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348.

A still further aspect of the present invention features antibodies andantibody preparations specifically reactive with an epitope of theDelta3 protein. In preferred embodiments, the antibody specificallybinds to an epitope of a polypeptide shown in SEQ ID NOs: 2, 25, 28, 30,32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded bythe cDNA of a clone deposited with the ATCC® as Accession Number 98348.

In yet a further aspect, the invention provides substantially purifiedantibodies or fragments thereof, including human or non-human antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NOs: 2, 25, 28, 30, 32,34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC® as Accession Number98348; a fragment of at least 15 amino acid residues of the amino acidsequence of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46,or the amino acid sequence encoded by the cDNA of a clone deposited withthe ATCC® as Accession Number 98348; an amino acid sequence which is atleast 95% identical to the amino acid sequence of SEQ ID NOs: 2, 25, 28,30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encodedby the cDNA of a clone deposited with the ATCC® as Accession Number98348 wherein the percent identity is determined using the ALIGN programof the GCG software package with a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4; and an amino acid sequencewhich is encoded by a nucleic acid molecule which hybridizes to thenucleic acid molecule consisting of SEQ ID NOs: 1, 3, 24, 26, 27, 29,31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence of the cDNAof a clone deposited with the ATCC® as Accession Number 98348, underconditions of hybridization of 6×SSC at 45□C. and washing in 0.2×SSC,0.1% SDS at 65□C. In various embodiments, the substantially purifiedantibodies of the invention, or fragments thereof, can be human,non-human, chimeric and/or humanized antibodies.

In another aspect, the invention provides human or non-human antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NOs: 2, 25, 28, 30, 32,34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC® as Accession Number98348; a fragment of at least 15 amino acid residues of the amino acidsequence of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46,or the amino acid sequence encoded by the cDNA of a clone deposited withthe ATCC® as Accession Number 98348; an amino acid sequence which is atleast 95% identical to the amino acid sequence of SEQ ID NOs: 2, 25, 28,30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encodedby the cDNA of a clone deposited with the ATCC® as Accession Number98348 wherein the percent identity is determined using the ALIGN programof the GCG software package with a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4; and an amino acid sequencewhich is encoded by a nucleic acid molecule which hybridizes to thenucleic acid molecule consisting of SEQ ID NOs: 1, 3, 24, 26, 27, 29,31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence of the cDNAof a clone deposited with the ATCC® as Accession Number 98348, underconditions of hybridization of 6×SSC at 45° C. and washing in 0.2×SSC,0.1% SDS at 65° C. With respect to non-human antibodies, such antibodiescan be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.Alternatively, the non-human antibodies of the invention can be chimericand/or humanized antibodies. In addition, the human and non-humanantibodies of the invention can be polyclonal antibodies or monoclonalantibodies.

In still a further aspect, the invention provides monoclonal antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NOs: 2, 25, 28, 30, 32,34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC® as Accession Number98348; a fragment of at least 15 amino acid residues of the amino acidsequence of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46;an amino acid sequence which is at least 95% identical to the amino acidsequence of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46,or the amino acid sequence encoded by the cDNA of a clone deposited withthe ATCC® as Accession Number 98348, wherein the percent identity isdetermined using the ALIGN program of the GCG software package with aPAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4; and an amino acid sequence which is encoded by a nucleicacid molecule which hybridizes to the nucleic acid molecule consistingof SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45,or the nucleotide sequence of the cDNA of a clone deposited with theATCC® as Accession Number 98348, under conditions of hybridization of6×SSC at 45° C. and washing in 0.2×SSC, 0.1% SDS at 65° C. Themonoclonal antibodies can be human, humanized, chimeric and/or non-humanantibodies.

In a particularly preferred embodiment, the substantially purifiedantibodies or fragments thereof, the human and non-human antibodies orfragments thereof, and/or monoclonal antibodies or fragments thereof, ofthe invention specifically bind to an extracellular domain of the aminoacid sequence of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348. Preferably, theextracellular domain to which the antibody, or fragment thereof, bindscomprises amino acid residues 1-529 of SEQ ID NO: 2 of human Delta3, oramino acid residues 1-530 of SEQ ID NO: 25 of murine Delta3.

Any of the antibodies of the invention can be conjugated to atherapeutic moiety or to a detectable substance. Non-limiting examplesof detectable substances that can be conjugated to the antibodies of theinvention are an enzyme, a prosthetic group, a fluorescent material, aluminescent material, a bioluminescent material, and a radioactivematerial.

The invention also provides a kit containing an antibody of theinvention and instructions for use. Such kits can also comprise anantibody of the invention conjugated to a detectable substance andinstructions for use. Still another aspect of the invention is apharmaceutical composition comprising an antibody of the invention and apharmaceutically acceptable carrier. In preferred embodiments, thepharmaceutical composition contains an antibody of the invention, atherapeutic moiety, and a pharmaceutically acceptable carrier.

In addition, the polypeptides of the invention or biologically activeportions thereof, or antibodies of the invention, can be incorporatedinto pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

The invention also features transgenic non-human animals which include(and preferably express) a heterologous form of a Delta3 gene describedherein, or which misexpress (e.g., do not express) an endogenous Delta3gene (e.g., an animal in which expression of one or more of the subjectDelta3 proteins is disrupted). Such a transgenic animal can serve as ananimal model for studying cellular or tissue disorders comprisingmutated or mis-expressed Delta3 alleles or for use in drug screening.For example, the transgenic animals of the invention can be used as ananimal model to study a neurological disease, e.g., ACCPN.Alternatively, such a transgenic animal can be useful for expressingrecombinant polypeptides, and for generating cells, e.g., cell linesthat can, for example, be utilized as part of screening techniques.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a polypeptide of theinvention. In general, such methods entail measuring a biologicalactivity of the polypeptide in the presence and absence of a testcompound and identifying those compounds which alter the activity of thepolypeptide.

The invention also features methods for identifying a compound whichmodulates the expression of a polypeptide or nucleic acid of theinvention by measuring the expression of the polypeptide or nucleic acidin the presence and absence of the compound.

In one embodiment, the invention provides assays, e.g., for screeningtest compounds to identify agonists, or alternatively, antagonists, of aDelta3 activity. For example, the test compound may positively ornegatively influence an interaction between a Delta3 protein and aDelta3 target molecule, for example, a Notch protein. An exemplarymethod includes the steps of (i) combining a Delta3 polypeptide oractive fragment thereof, with a Delta3 target molecule, e.g., Notch, anda test compound, e.g., under conditions wherein, but for the testcompound, the Delta3 protein and target molecule are able to interact;and (ii) detecting the formation of a complex which includes the Delta3protein and the target molecule either by directly quantitating thecomplex, by measuring inductive effects of the Delta3 protein, or, inthe instance of a substrate, measuring the conversion to product. Astatistically significant change, such as a decrease, in the interactionof the Delta3 and target molecule in the presence of a test compound(relative to what is detected in the absence of the test compound) isindicative of a modulation (e.g., suppression or potentiation of theinteraction between the Delta3 protein and the target molecule).

The invention provides yet other methods for identifying compounds whichmodulate a Delta activity. For example, a compound that interacts with aDelta3 protein can be identified by contacting a Delta3 protein with atest compound. Either the test compound or the Delta3 protein can belabeled. Optionally, the non-labeled compound or Delta3 protein can beattached to a solid phase support. Binding of the test compound to theDelta3 protein can then be determined, e.g by measuring the amount oflabel. Such a Delta3 binding molecule can be an agonist or anantagonist. In one embodiment, an agonist of a Delta3 activity isidentified by contacting a cell having a Delta3 protein with a testcompound and measuring a Delta3 activity, e.g., expression of a genewhich is regulated by binding of a protein, e.g., a Notch protein, toDelta3. An increased expression of the gene when the cell is incubatedwith the test compound relative to incubation in the absence of the testcompound indicates that the test compound is a Delta3 agonist. The genethat is monitored can also be a reporter gene transfected to a cell, thereporter gene being under the control of a promoter of a gene which isregulated by Delta3.

In another aspect, the invention provides methods for modulatingactivity of a polypeptide of the invention comprising contacting a cellwith an agent that modulates (inhibits or stimulates) the activity orexpression of a polypeptide of the invention such that activity orexpression in the cell is modulated. In one embodiment, the agent is anantibody that specifically binds to a polypeptide of the invention.

In another embodiment, the agent modulates expression of a polypeptideof the invention by modulating transcription, splicing, or translationof an mRNA encoding a polypeptide of the invention. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of an mRNA encoding apolypeptide of the invention.

Yet another aspect of the present invention concerns methods fortreating diseases or conditions that are caused or contributed to by anaberrant Delta3 expression, level, or activity, e.g., aberrant cellproliferation, degeneration or differentiation, in a subject, byadministering to the subject an effective amount of a modulator (e.g.,an agonist or antagonist) of a Delta3 activity. In one embodiment, anagonist or antagonist can modulate Delta3 protein levels, by, e.g.,modulating expression of a Delta3 gene. A modulator can, for example, bea protein of the invention, or, alternatively, a nucleic acid of theinvention. In other embodiments, the modulator is a peptide, antibody,peptidomimetic, or other small organic molecule. For example,administration of a therapeutic comprised of a Delta3 agonist can beuseful for promoting the tissue regeneration or repair needed toeffectively treat a nerve injury, neurodegenerative disease, orneurodevelopmental disorder including but not limited to peripheralneuropathies, e.g., ACCPN, stroke, dementia, Alzheimer's disease,Parkinson's disease, Huntington's chorea, amylotrophic lateralsclerosis, and the like, as well as spinocerebellar degenerations.

Alternatively, administration of a Delta3 antagonist may be toeffectively treat a neoplastic or hyperplastic disease, particularly ofendothelial tissue.

Additionally, Delta3 agonists or antagonists may be used to treatvarious hematologic abnormalities such as neutropenia seen in patientsundergoing chemotherapy, or immunodeficiency disorders such as AIDS.Delta3 nucleic acids, polypeptides or modulators thereof can also beutilized in treating or ameliorating a symptom of obesity and/ordisorders that accompany or are exacerbated by an obese state, such ascardiovascular and circulatory disorders, metabolic abnormalitiestypical of obesity, such as hyperinsulinemia, insulin resistance,diabetes, including non-insulin dependent diabetes mellitus (NIDDM),insulin dependent diabetes mellitus (IDDM), and maturity onset diabetesof the young (MODY), disorders of energy homeostasis, disordersassociated with lipid metabolism, such as cachexia, disorders associatedwith abnormal vasculogenesis (e.g., cancers, including, but not limitedto, cancers of the epithelia (e.g., carcinomas of the pancreas, stomach,liver, secretory glands (e.g., adenocarcinoma), bladder, lung, breast,skin (e.g., fibromatosis or malignant melanoma), reproductive tractincluding prostate gland, ovary, cervix and uterus); cancers of thehematopoietic and immune system (e.g., leukemias and lymphomas); cancersof the central nervous, brain system and eye (e.g., gliomas,neuroblastoma and retinoblastoma); and cancers of connective tissues,bone, muscles and vasculature (e.g., hemangiomas and sarcomas)),disorders related to fetal development, in particular, disordersinvolving development of lung and kidney, lung-related disorders,atherosclerosis, e.g., the initiation and progression ofatherosclerosis; and inflammatory-related disorders, e.g., asthma,allergy, and autoimmune disorders, neurological disorders, includingdevelopmental, cognitive and personality-related disorders, renaldisorders, adrenal gland-related disorders; and disorders relating toskeletal muscle, such as dystrophic disorders.

The invention also provides methods for treating diseases or conditionsassociated with one or more specific Delta alleles, e.g., mutant allele,comprising administering to the subject an effective amount of atherapeutic compound. In one embodiment, the therapeutic compound iscapable of compensating for the effect of the specific Delta allele. Inanother embodiment, the therapeutic compound is capable of modulating aDelta3 activity. In a one embodiment, the Delta allele is a Delta3allele. For example, in one embodiment, the disease or condition is aneurological disease, e.g., ACCPN.

A further aspect of the present invention provides a method fordetermining whether a subject is at risk for developing a disease orcondition, which is caused by or contributed to by an aberrant Delta3activity, e.g. aberrant cell proliferation, degeneration ordifferentiation. In one embodiment, the disease or condition is aneurological disease, e.g., ACCPN. The method includes detecting, in atissue of the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding aDelta3 protein, e.g., as shown in SEQ ID Nos: 1, 3, 24, 26, 27, 29, 31,33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence of the cDNA ofa clone deposited with the ATCC® as Accession Number 98348, or a homologthereof; or (ii) the mis-expression of a Delta3 gene. In oneembodiments, detecting the genetic lesion includes ascertaining theexistence of at least one of the following: a deletion of one or morenucleotides from a Delta3 gene; an addition of one or more nucleotidesto the gene, a substitution of one or more nucleotides of the gene, agross chromosomal rearrangement of the gene; an alteration in the levelof a messenger RNA transcript of the gene; the presence of a non-wildtype splicing pattern of a messenger RNA transcript of the gene; and/ora non-wild type level of the protein.

In a preferred embodiment, the invention provides a method fordetermining whether a subject has or is at risk of developing a diseaseor condition associated with a specific Delta3 allele, comprisingdetermining the identity of a Delta3 allele in the subject. In an evenmore preferred embodiment, the disease or condition is a neurologicaldisease, e.g., ACCPN. In another preferred embodiment, the disease is avascular disorder. In another preferred embodiment, the disease is aneoplastic disorder. In another preferred embodiment the disease is ahematologic disorder. In another preferred embodiment the disease is animmunodeficiency disorder.

For example, detecting the genetic lesion or determining the identity ofa Delta allele, e.g., a Delta3 allele, can include: (i) providing aprobe/primer comprised of an oligonucleotide which hybridizes to a senseor antisense sequence of a Delta3 gene or naturally-occurring mutantsthereof, or 5′ or 3′ flanking sequences naturally associated with theDelta3 gene; (ii) contacting the probe/primer with an appropriatenucleic acid containing sample; and (iii) detecting, by hybridization ofthe probe/primer to the nucleic acid, the presence or absence of thegenetic lesion, e.g., by utilizing the probe/primer to determine thenucleotide sequence of the Delta3 gene and, optionally, of the flankingnucleic acid sequences. For instance, the primer can be employed in apolymerase chain reaction (PCR) or in a ligation chain reaction (LCR).

In another diagnostic method of the invention, at least a portion of aDelta3 gene of a subject is sequenced and the nucleotide sequence iscompared to that of a wild-type Delta3 gene, to determine the presenceof a genetic lesion. Another preferred diagnostic method of theinvention is single strand conformation polymorphism (SSCP) whichdetects differences in electrophoretic mobility between mutant andwild-type nucleic acids.

In alternate embodiments, the diagnostic methods comprise determiningthe level of a Delta3 protein in an immunoassay using an antibody whichis specifically immunoreactive with a wildtype or mutant Delta3 protein.

In another aspect, the present invention provides methods for detectingthe presence of the activity or expression of a polypeptide of theinvention in a biological sample by contacting the biological samplewith an agent capable of detecting an indicator of activity such thatthe presence of activity is detected in the biological sample.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a polypeptide of the invention, (ii) mis-regulation of a geneencoding a polypeptide of the invention, and (iii) aberrantpost-translational modification of a polypeptide of the inventionwherein a wild-type form of the gene encodes a polypeptide having theactivity of the polypeptide of the invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

FTHMA-070 and TANGO85

The present invention is based, at least in part, on the discovery of agene encoding FTHMA-070, a protein having homology to tumor necrosisfactor (TNF) receptor and on the discovery of a gene encoding T85 (alsoreferred to as FMHB-SD4 or FMHB-6D4).

The FTHMA-070 cDNA described below (SEQ ID NO:53) has a 1203 nucleotideopen reading frame (nucleotides 73-1275 of SEQ ID NO:53; SEQ ID NO:55)which encodes a 403 amino acid protein (SEQ ID NO:54). This proteinincludes a predicted signal sequence of about 21 amino acids (from aminoacid 1 to about amino acid 21 of SEQ ID NO:54) and a predicted matureprotein of about 382 amino acids (from about amino acid 22 to amino acid403 of SEQ ID NO:54; SEQ ID NO:56).

The T85 cDNA described below (SEQ ID NO:57) has a 2259 nucleotide openreading frame (nucleotides 958-3216 of SEQ ID NO:57; SEQ ID NO:59) whichencodes a 753 amino acid protein (SEQ ID NO:58). This protein includes apredicted signal sequence of about 20 amino acids (from amino acid 1 toabout amino acid 20 of SEQ ID NO:58) and a predicted mature protein ofabout 733 amino acids (from about amino acid 21 to amino acid 753 of SEQID NO:58; SEQ ID NO:60).

The nucleic acid and polypeptide molecules of the present invention areuseful as modulating agents in regulating a variety of cellularprocesses. Accordingly, in one aspect, this invention provides isolatednucleic acid molecules encoding FTHMA-070 proteins or biologicallyactive portions thereof, as well as nucleic acid fragments suitable asprimers or hybridization probes for the detection of FTHMA-070-encodingnucleic acids. In another aspect, this invention provides isolatednucleic acid molecules encoding T85 proteins or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of T85-encoding nucleic acids.

The invention features a nucleic acid molecule which is at least 45% (or55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequenceshown in SEQ ID NO:53, or SEQ ID NO:55, or a complement thereof. Theinvention features a nucleic acid molecule which includes a fragment ofat least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, or 1290) nucleotides of the nucleotide sequence shown inSEQ ID NO:53, or SEQ ID NO:55, or a complement thereof.

The invention also features a nucleic acid molecule which includes anucleotide sequence encoding a protein having an amino acid sequencethat is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical tothe amino acid sequence of SEQ ID NO:54, SEQ ID NO:56. In a preferredembodiment, a FTHMA-070 nucleic acid molecule has the nucleotidesequence shown SEQ ID NO:53, or SEQ ID NO:55.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:54or SEQ ID NO:56, the fragment including at least 15 (25, 30, 50, 100,150, 300, or 400) contiguous amino acids of SEQ ID NO:54 or SEQ IDNO:56.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:54 or SEQ ID NO:56, wherein the nucleic acidmolecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:53or SEQ ID NO:55 under stringent conditions.

Also within the invention are: an isolated FTHMA-070 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:56 (maturehuman FTHMA-070) or the amino acid sequence of SEQ ID NO:54 (immaturehuman FTHMA-070).

Also within the invention are: an isolated FTHMA-070 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:55; and an isolated FTHMA-070 protein which is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:55.

Also within the invention is a polypeptide which is a naturallyoccurring allelic variant of a polypeptide that includes the amino acidsequence of SEQ ID NO:54 or SEQ ID NO:56, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:53 or SEQ ID NO:55 under stringentconditions.

Another embodiment of the invention features FTHMA-070 nucleic acidmolecules which specifically detect FTHMA-070 nucleic acid molecules.For example, in one embodiment, a FTHMA-070 nucleic acid moleculehybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:53, SEQ ID NO:55, or acomplement thereof. In another embodiment, the FTHMA-070 nucleic acidmolecule is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600,650, 700, 800, 900, 1000, or 1200) nucleotides in length and hybridizesunder stringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:53 or SEQ ID NO:55, or acomplement thereof. In another embodiment, the invention provides anisolated nucleic acid molecule which is antisense to the coding strandof a FTHMA-070 nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a FTHMA-070 nucleic acid molecule of theinvention. In another embodiment the invention provides a host cellcontaining such a vector. The invention also provides a method forproducing FTHMA-070 protein by culturing, in a suitable medium, a hostcell of the invention containing a recombinant expression vector suchthat a FTHMA-070 protein is produced.

Another aspect of this invention features isolated or recombinantFTHMA-070 proteins and polypeptides. Preferred FTHMA-070 proteins andpolypeptides possess at least one biological activity possessed bynaturally occurring human FTHMA-070, e.g., the ability to formprotein:protein interactions with other proteins.

The FTHMA-070 proteins of the present invention, or biologically activeportions thereof, can be operatively linked to a non-FTHMA-070polypeptide (e.g., heterologous amino acid sequences) to form FTHMA-070fusion proteins. The invention further features antibodies thatspecifically bind FTHMA-070 proteins, such as monoclonal or polyclonalantibodies. In addition, the FTHMA-070 proteins or biologically activeportions thereof can be incorporated into pharmaceutical compositions,which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of FTHMA-070 activity or expression in a biological sampleby contacting the biological sample with an agent capable of detectingan indicator of FTHMA-070 activity such that the presence of FTHMA-070activity is detected in the biological sample.

In another aspect, the invention provides a method for modulatingFTHMA-070 activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates) FTHMA-070 activity or expression suchthat FTHMA-070 activity or expression in the cell is modulated. In oneembodiment, the agent is an antibody that specifically binds toFTHMA-070 protein. In another embodiment, the agent modulates expressionof FTHMA-070 by modulating transcription of a FTHMA-070 gene, splicingof a FTHMA-070 mRNA, or translation of a FTHMA-070 mRNA. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of the FTHMA-070 mRNA orthe FTHMA-070 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant FTHMA-070protein or nucleic acid expression or activity by administering an agentwhich is a FTHMA-070 modulator to the subject. In one embodiment, theFTHMA-070 modulator is a FTHMA-070 protein. In another embodiment theFTHMA-070 modulator is a FTHMA-070 nucleic acid molecule. In otherembodiments, the FTHMA-070 modulator is a peptide, peptidomimetic, orother small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a FTHMA-070 protein; (ii) mis-regulation of a gene encoding aFTHMA-070 protein; and (iii) aberrant post-translational modification ofa FTHMA-070 protein, wherein a wild-type form of the gene encodes aprotein with a FTHMA-070 activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a FTHMA-070 protein.In general, such methods entail measuring a biological activity of aFTHMA-070 protein in the presence and absence of a test compound andidentifying those compounds which alter the activity of the FTHMA-070protein.

The invention also features methods for identifying a compound whichmodulates the expression of FTHMA-070 by measuring the expression ofFTHMA-070 in the presence and absence of a compound.

The invention features a nucleic acid molecule which is at least 45% (or55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequenceshown in SEQ ID NO:57, or SEQ ID NO:59, or a complement thereof. Theinvention features a nucleic acid molecule which includes a fragment ofat least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, or 1290) nucleotides of the nucleotide sequence shown inSEQ ID NO:57, or SEQ ID NO:59, or a complement thereof.

The invention also features a nucleic acid molecule which includes anucleotide sequence encoding a protein having an amino acid sequencethat is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical tothe amino acid sequence of SEQ ID NO:58 or SEQ ID NO:60. In a preferredembodiment, a T85 nucleic acid molecule has the nucleotide sequenceshown SEQ ID NO:57, or SEQ ID NO:59.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:58or SEQ ID NO:60, the fragment including at least 15 (25, 30, 50, 100,150, 300, or 400) contiguous amino acids of SEQ ID NO:58 or SEQ IDNO:60.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:58 or SEQ ID NO:60, wherein the nucleic acidmolecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:57or SEQ ID NO:59 under stringent conditions.

Also within the invention are: an isolated T85 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the amino acid sequence of SEQ ID NO:60 (mature humanT85) or the amino acid sequence of SEQ ID NO:58 (immature human T85);and an isolated T85 protein having an amino acid sequence that is atleast about 85%, 95%, or 98% identical to one or more of the fibronectintype III domains and Ig superfamily domains described herein.

Also within the invention are: an isolated T85 protein which is encodedby a nucleic acid molecule having a nucleotide sequence that is at leastabout 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:59; anisolated T85 protein which is encoded by a nucleic acid molecule havinga nucleotide sequence at least about 65% preferably 75%, 85%, or 95%identical a fibronectin III or Ig superfamily domain encoding portion ofSEQ ID NO:57; and an isolated T85 protein which is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:59.

Also within the invention is a polypeptide which is a naturallyoccurring allelic variant of a polypeptide that includes the amino acidsequence of SEQ ID NO:58 or SEQ ID NO:60, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:57 or SEQ ID NO:59 under stringentconditions.

Another embodiment of the invention features T85 nucleic acid moleculeswhich specifically detect T85 nucleic acid molecules. For example, inone embodiment, a T85 nucleic acid molecule hybridizes under stringentconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:57 or SEQ ID NO:59, or a complement thereof. In anotherembodiment, the T85 nucleic acid molecule is at least 300 (325, 350,375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1290)nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising the nucleotide sequence shown in SEQ IDNO:57 or SEQ ID NO:59, or a complement thereof. In a preferredembodiment, an isolated T85 nucleic acid molecule comprises a nucleotidesequence of SEQ ID NO:57 which encodes a fibronectin type III domain, ora complement thereof. In another preferred embodiment, an isolated T85nucleic acid molecule comprises a nucleotide of SEQ ID NO:57 whichencodes an Ig superfamily domain, or a complement thereof. In anotherembodiment, the invention provides an isolated nucleic acid moleculewhich is antisense to the coding strand of a T85 nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a T85 nucleic acid molecule of theinvention. In another embodiment the invention provides a host cellcontaining such a vector. The invention also provides a method forproducing T85 protein by culturing, in a suitable medium, a host cell ofthe invention containing a recombinant expression vector such that a T85protein is produced.

Another aspect of this invention features isolated or recombinant T85proteins and polypeptides. Preferred T85 proteins and polypeptidespossess at least one biological activity possessed by naturallyoccurring human T85, e.g., the ability to form protein:proteininteractions with other proteins.

The T85 proteins of the present invention, or biologically activeportions thereof, can be operatively linked to a non-T85 polypeptide(e.g., heterologous amino acid sequences) to form T85 fusion proteins.The invention further features antibodies that specifically bind T85proteins, such as monoclonal or polyclonal antibodies. In addition, theT85 proteins or biologically active portions thereof can be incorporatedinto pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of T85 activity or expression in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of T85 activity such that the presence of T85 activity isdetected in the biological sample.

In another aspect, the invention provides a method for modulating T85activity comprising contacting a cell with an agent that modulates(inhibits or stimulates) T85 activity or expression such that T85activity or expression in the cell is modulated. In one embodiment, theagent is an antibody that specifically binds to T85 protein. In anotherembodiment, the agent modulates expression of T85 by modulatingtranscription of a T85 gene, splicing of a T85 mRNA, or translation of aT85 mRNA. In yet another embodiment, the agent is a nucleic acidmolecule having a nucleotide sequence that is antisense to the codingstrand of the T85 mRNA or the T85 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant T85 proteinor nucleic acid expression or activity by administering an agent whichis a T85 modulator to the subject. In one embodiment, the T85 modulatoris a T85 protein. In another embodiment the T85 modulator is a T85nucleic acid molecule. In other embodiments, the T85 modulator is apeptide, peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a T85 protein; (ii) mis-regulation of a gene encoding a T85protein; and (iii) aberrant post-translational modification of a T85protein, wherein a wild-type form of the gene encodes a protein with aT85 activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a T85 protein. Ingeneral, such methods entail measuring a biological activity of a T85protein in the presence and absence of a test compound and identifyingthose compounds which alter the activity of the T85 protein.

The invention also features methods for identifying a compound whichmodulates the expression of T85 by measuring the expression of T85 inthe presence and absence of a compound.

TANGO 77

The present invention is based, at least in part, on the discovery of agene encoding Tango-77, a secreted protein that is predicted to be amember of the cytokine superfamily. The Tango-77 cDNA described below(SEQ ID NO:71) has three possible open reading frames. The firstpotential open reading frame encompasses 534 nucleotides extending fromnucleotide 356 to nucleotide 889 of SEQ ID NO:71 (SEQ ID NO:73) andencodes a 178 amino acid protein (SEQ ID NO:72). This protein mayinclude a predicted signal sequence of about 63 amino acids (from aboutamino acid 1 to about amino acid 63 of SEQ ID NO:72 (SEQ ID NO:74) and apredicted mature protein of about 115 amino acids (from about amino acid64 to amino acid 178 of SEQ ID NO:72 (SEQ ID NO:75)).

The second potential open reading frame encompasses 498 nucleotidesextending from nucleotide 389 to nucleotide 889 of SEQ ID NO:71 (SEQ IDNO:76) and encodes a 167 amino acid protein (SEQ ID NO:77). This proteinmay include a predicted signal sequence of about 52 amino acids (fromabout amino acid 1 to about amino acid 52 of SEQ ID NO:77 (SEQ IDNO:78)) and a predicted mature protein of about 115 amino acids (fromabout amino acid 52 to amino acid 167 of SEQ ID NO:77 (SEQ ID NO:79)).

The third potential open reading frame encompasses 408 nucleotidesextending from nucleotide 481 to nucleotide 889 of SEQ ID NO:71 (SEQ IDNO:80) and encodes a 136 amino acid protein (SEQ ID NO:81). This proteinincludes a predicted signal sequence of about 21 amino acids (from aboutamino acid 1 to about amino acid 21 of SEQ ID NO:81 (SEQ ID NO:82)) anda predicted mature protein of about 115 amino acids (from about aminoacid 22 to amino acid 136 of SEQ ID NO:81 (SEQ ID NO:83)).

As used herein, the terms “Tango-77”, “Tango-77 protein”, “Tango-77polypeptide” and the like, can refer and polypeptide produced by thecDNA of SEQ ID NO:71 including any and all of the Tango-77 gene productsdescribed above.

Tango-77 is expected to inhibit inflammation and play a functional rolesimilar to that of secreted IL-1ra. For example, it is expected thatTango-77 may bind to the IL-1 receptor, thus blocking receptoractivation by inhibiting the binding of IL-1α and IL-1β to the receptor.Alternatively, Tango-77 may inhibit inflammation through anotherpathway, for example, by binding to a novel receptor. Accordingly,Tango-77 may be useful as a modulating agent in regulating a variety ofcellular processes including acute and chronic inflammation, e.g.,asthma, chronic myelogenous leukemia, rheumatoid arthritis, psoriasisand inflammatory bowel disease.

In one aspect, the invention provides isolated nucleic acid moleculesencoding Tango-77 or biologically active portions thereof, as well asnucleic acid fragments suitable as primers or hybridization probes forthe detection of Tango-77.

The invention encompasses methods of diagnosing and treating patientswho are suffering from a disorder associated with an abnormal level(undesirably high or undesirably low) of inflammation, abnormal activityof the IL-1 receptor complex, or abnormal activity of IL-1, byadministering a compound that modulates the expression of Tango-77 (atthe DNA, mRNA or protein level, e.g., by altering mRNA splicing) or byaltering the activity of Tango-77. Examples of such compounds includesmall molecules, antisense nucleic acid molecules, ribozymes, andpolypeptides.

The invention features a nucleic acid molecule which is at least 45%(e.g., 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotidesequence shown in SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ IDNO:80, the nucleotide sequence of the cDNA insert of the plasmiddeposited with ATCC as Accession Number (the “cDNA of ATCC 98807”), or acomplement thereof.

The invention features a nucleic acid molecule which includes a fragmentof at least 100 (e.g., 250, 325, 350, 375, 400, 425, 450, 500, 550, 600,650, 700, 800, 900, or 989) nucleotides of the nucleotide sequence shownin SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:80, thenucleotide sequence of the cDNA ATCC 98807, or a complement thereof.

The invention also features a nucleic acid molecule which includes anucleotide sequence encoding a protein having an amino acid sequencethat is at least 45% (55%, 65%, 75%, 85%, 95%, or 98%) identical to theamino acid sequence of SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, or the amino acid sequence encoded bythe cDNA of ATCC 98807.

In a preferred embodiment, a Tango-77 nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76,SEQ ID NO:80 or the nucleotide sequence of the cDNA of ATCC 98807.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ IDNO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, wherein the fragmentincludes at least 15 (e.g., 25, 30, 50, 100, 150, or 178) contiguousamino acids of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77,SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, orthe polypeptide encoded by the cDNA of ATCC Accession Number 98807.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or anamino acid sequence encoded by the cDNA of ATCC Accession Number 98807,wherein the nucleic acid molecule hybridizes to a nucleic acid moleculecomprising SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:80, or acomplement thereof under stringent conditions.

Also within the invention are: an isolated Tango-77 protein having anamino acid sequence that is at least about 45%, preferably 65%, 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:75,SEQ ID NO:79 or SEQ ID NO:83 (mature human Tango-77), or the amino acidsequence of SEQ ID NO:72, SEQ ID NO:77 or SEQ ID NO:81 (immature humanTango-77).

Also within the invention are: an isolated Tango-77 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:73, SEQ ID NO:76, SEQ ID NO:80 or the cDNA of ATCC 98807; and anisolated Tango-77 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:80, thenon-coding strand of the cDNA of ATCC 98807, or the complement thereof.

Also within the invention is a polypeptide which is a naturallyoccurring allelic variant of a polypeptide that includes the amino acidsequence of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, or anamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC as Accession Number 98807, wherein the polypeptide is encodedby a nucleic acid molecule which hybridizes to a nucleic acid moleculecomprising SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:80 or thecomplement thereof under stringent conditions.

Another embodiment of the invention features Tango-77 nucleic acidmolecules which specifically detect Tango-77 nucleic acid moleculesrelative to nucleic acid molecules encoding other members of thecytokine superfamily. For example, in one embodiment, a Tango-77 nucleicacid molecule hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:76, SEQ ID NO:80, the cDNA of ATCC 98807, or acomplement thereof. In another embodiment, the Tango-77 nucleic acidmolecule is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600,650, 700, 800, 900, or 989) nucleotides in length and hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76,SEQ ID NO:80, the cDNA of ATCC 98807, or a complement thereof. In yetanother embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a Tango-77 nucleicacid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a Tango-77 nucleic acid molecule of theinvention. In another embodiment, the invention provides a host cellcontaining such a vector. The invention also provides a method forproducing Tango-77 protein by culturing, in a suitable medium, a hostcell of the invention containing a recombinant expression vector suchthat a Tango-77 protein is produced.

Another aspect of this invention features isolated or recombinantTango-77 proteins and polypeptides. Preferred Tango-77 proteins andpolypeptides possess at least one biological activity possessed bynaturally occurring human Tango-77, e.g., (i) the ability to interactwith proteins in the Tango-77 signalling pathway (ii) the ability tointeract with a Tango-77 ligand or receptor; or (iii) the ability tointeract with an intracellular target protein, (iv) the ability tointeract with a protein involved in inflammation and (v) the ability tobind the IL-1 receptor. Other activities include the induction andsuppression of polypeptide interleukins, cytokines and growth factors.

The Tango-77 proteins of the present invention, or biologically activeportions thereof, can be operably linked to a non-Tango-77 polypeptide(e.g., heterologous amino acid sequences) to form Tango-77 fusionproteins. The invention further features antibodies that specificallybind Tango-77 proteins, such as monoclonal or polyclonal antibodies. Inaddition, the Tango-77 proteins or biologically active portions thereofcan be incorporated into pharmaceutical compositions, which optionallyinclude pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of Tango-77 activity or expression in a biological sampleby contacting the biological sample with an agent capable of detectingan indicator of Tango-77 activity or expression such that the presenceof Tango-77 activity or expression is detected in the biological sample.

In another aspect, the invention provides a method for modulatingTango-77 activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates)

Tango-77 activity or expression such that Tango-77 activity orexpression in the cell is modulated. In one embodiment, the agent is anantibody that specifically binds to Tango-77 protein. In anotherembodiment, the agent modulates expression of Tango-77 by modulatingtranscription of a Tango-77 gene, splicing of a Tango-77 mRNA, ortranslation of a Tango-77 mRNA. In yet another embodiment, the agent isa nucleic acid molecule having a nucleotide sequence that is antisenseto the coding strand of the Tango-77 mRNA or the Tango-77 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant Tango-77protein activity or nucleic acid expression by administering an agentwhich is a Tango-77 modulator to the subject. In one embodiment, theTango-77 modulator is a Tango-77 protein. In another embodiment, theTango-77 modulator is a Tango-77 nucleic acid molecule. In otherembodiments, the Tango-77 modulator is a peptide, peptidomimetic, orother small molecule. In a preferred embodiment, the disordercharacterized by aberrant Tango-77 protein or nucleic acid expressioncan include chronic and acute inflammation.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a Tango-77 protein; (ii) mis-regulation of a gene encoding aTango-77 protein; and (iii) aberrant post-translational modification ofa Tango-77 protein, wherein a wild-type form of the gene encodes aprotein with a Tango-77 activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a Tango-77 protein.In general, such methods entail measuring a biological activity of aTango-77 protein in the presence and absence of a test compound andidentifying those compounds which alter the activity of the Tango-77protein.

The invention also features methods for identifying a compound whichmodulates the expression of Tango-77 by measuring the expression ofTango-77 in the presence and absence of a compound.

SPOIL

The present invention is based, at least in part, on the discovery ofnovel nucleic acid molecules which encode a novel family of proteinshaving homology to IL-1 receptor antagonist (IL-1ra) molecules, referredto herein as SPOIL nucleic acid and protein molecules. The SPOILmolecules of the present invention are useful as modulating agents inregulating a variety of cellular processes. Accordingly, in one aspect,this invention provides isolated nucleic acid molecules encoding SPOILproteins and biologically active portions thereof, as well as nucleicacid fragments suitable as primers or hybridization probes for thedetection of SPOIL-encoding nucleic acids. In one embodiment, anisolated nucleic acid molecule of the present invention preferablyencodes a SPOIL protein which includes an interleukin-1 (IL-1) signaturedomain.

In another embodiment, an isolated nucleic acid molecule of the presentinvention preferably encodes a SPOIL protein which includes a SPOILsignature motif. In another embodiment, an isolated nucleic acidmolecule of the present invention preferably encodes a SPOIL proteinwhich includes a SPOIL unique domain. In another embodiment, an isolatednucleic acid molecule of the present invention preferably encodes aSPOIL protein which includes a SPOIL C-terminal unique domain. Inanother embodiment, an isolated nucleic acid molecule of the presentinvention preferably encodes a SPOIL protein which includes a signalsequence and/or is secreted. In yet another embodiment, an isolatednucleic acid molecule of the present invention preferably encodes aSPOIL protein which lacks a signal sequence and/or is intracellular. Inanother embodiment, the nucleic acid molecule is a naturally occurringnucleotide sequence.

In another embodiment, a nucleic acid molecule of the invention has 65%identity with the nucleotide sequence shown in SEQ ID NO:89, SEQ IDNO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, the DNA insert of theplasmid deposited with ATCC as Accession Number 98984 or a complementthereof and, preferably, encodes a SPOIL protein. In yet anotherembodiment, the isolated nucleic acid molecule has 65% identity with thenucleotide sequence shown in SEQ ID NO:91, SEQ ID NO:103, SEQ ID NO:106,SEQ ID NO:114, or a complement thereof and, preferably, encodes a SPOILprotein. In a preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of a mammalian protein, (e.g., a humanor mouse SPOIL protein.)

In another embodiment, the isolated nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently homologous to the amino acid sequence of SEQ ID NO:90; SEQID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encodedby the DNA insert of the plasmid deposited with ATCC as Accession Number98883, or the DNA insert of the plasmid deposited with ATCC as AccessionNumber 98984 and, preferably, encodes a SPOIL protein. In a preferredembodiment, the nucleic acid molecule has the nucleotide sequence shownin SEQ ID NO:91, SEQ ID NO:103, SEQ ID NO:106, or SEQ ID NO:114. Inanother preferred embodiment, the nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:104,SEQ ID NO:112, the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the DNA insert of the plasmid deposited withATCC as Accession Number 98984.

Another embodiment of the invention features isolated nucleic acidmolecules which hybridize under stringent conditions to a nucleic acidmolecule consisting of nucleotides 135-428 or nucleotides 495-746 of SEQID NO:89. In yet another preferred embodiment, the isolated nucleic acidmolecules hybridize under stringent conditions to a nucleic acidmolecule consisting of nucleotides 1-280, 123-260, or nucleotides390-1291 of SEQ ID NO:101. In yet another preferred embodiment, theisolated nucleic acid molecules hybridize under stringent conditions toa nucleic acid molecule consisting of nucleotides 1-371, 98-721, ornucleotides 481-1377 of SEQ ID NO:104. In yet another preferredembodiment, the isolated nucleic acid molecules hybridize understringent conditions to a nucleic acid molecule consisting ofnucleotides 225-365, 96-575, or nucleotides 495-838 of SEQ ID NO:112. Inanother embodiment, the nucleic acid molecule is at least 300nucleotides in length. In another embodiment, the nucleic acid moleculeis at least 300 nucleotides in length and hybridizes under stringentconditions to a nucleic acid molecule comprising the nucleotide sequenceshown in SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:112, theDNA insert of the plasmid deposited with ATCC as Accession Number 98883,or the DNA insert of the plasmid deposited with ATCC as Accession Number98984, or a complement thereof. In yet another embodiment, the nucleicacid molecule is at least 300 nucleotides in length and encodes a SPOILprotein or portion thereof, preferably a biologically active portionthereof.

In a preferred embodiment, an isolated nucleic acid molecule comprisesnucleotides 135-428 of SEQ ID NO:89, or a complement thereof. In anotherembodiment, the nucleic acid molecule further comprises nucleotides1-134 of SEQ ID NO:89. In yet another embodiment, the nucleic acidmolecule further comprises nucleotides 429-746 of SEQ ID NO:89.

In another preferred embodiment, an isolated nucleic acid moleculecomprises nucleotides 124-630 of SEQ ID NO:101, or a complement thereof.In another embodiment, the nucleic acid molecule further comprisesnucleotides 1-123 of SEQ ID NO:101. In yet another embodiment, thenucleic acid molecule further comprises nucleotides 631-1291 of SEQ IDNO:101.

In another preferred embodiment, an isolated nucleic acid moleculecomprises nucleotides 98-721 of SEQ ID NO:104, or a complement thereof.In another embodiment, the nucleic acid molecule further comprisesnucleotides 1-97 of SEQ ID NO:104. In yet another embodiment, thenucleic acid molecule further comprises nucleotides 722-1377 of SEQ IDNO:104.

In another preferred embodiment, an isolated nucleic acid moleculecomprises nucleotides 96-575 of SEQ ID NO:112, or a complement thereof.In another embodiment, the nucleic acid molecule further comprisesnucleotides 1-95 of SEQ ID NO:112. In yet another embodiment, thenucleic acid molecule further comprises nucleotides 576-838 of SEQ IDNO:112.

Another embodiment the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a SPOIL nucleicacid.

Another aspect of the invention provides a vector comprising a nucleicacid molecule of the invention, preferably a SPOIL nucleic acidmolecule. In certain embodiments, the vector is a recombinant expressionvector. In another embodiment, the invention provides a host cellcontaining a vector of the invention. The invention also provides amethod for producing SPOIL protein by culturing in a suitable medium, ahost cell of the invention containing a recombinant expression vectorsuch that SPOIL protein is produced.

Another aspect of this invention features isolated or recombinantproteins and polypeptides, preferably SPOIL proteins or polypeptides. Inone embodiment, an isolated protein, preferably a SPOIL protein, has aSPOIL signature motif. In another embodiment, an isolated protein,preferably a SPOIL protein, has an IL-1 signature domain. In anotherembodiment, an isolated protein, preferably a SPOIL protein, has a SPOILunique domain. In another embodiment, an isolated protein, preferably aSPOIL protein, has a SPOIL C-terminal unique domain. In anotherembodiment, an isolated protein, preferably a SPOIL protein, has acombination of two or more of the above-stated domains and/or motifs. Inyet another embodiment, an isolated protein, preferably a SPOIL protein,has a signal sequence and/or is secreted. In yet another embodiment, anisolated protein, preferably a SPOIL protein, lacks a signal sequenceand/or is intracellular. In another embodiment, an isolated protein,preferably a SPOIL protein, has an amino acid sequence sufficientlyhomologous to the amino acid sequence of SEQ ID NO:90, SEQ ID NO:102,SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98883, orthe amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98984. This invention furtherfeatures isolated proteins, preferably SPOIL proteins, having an aminoacid sequence at least about 45% identical to a SPOIL unique domainamino acid sequence. This invention further features isolated proteins,preferably SPOIL proteins, having an amino acid sequence at least about45% identical to a SPOIL C-terminal unique domain amino acid sequence.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:90, SEQ ID NO:102, SEQ IDNO:105, SEQ ID NO:113, the amino acid sequence encoded by the DNA insertof the plasmid deposited with ATCC as Accession Number 98883, or theamino acid sequence encoded by the DNA insert of the plasmid depositedwith ATCC as Accession Number 98984, wherein the fragment comprises atleast 15 contiguous amino acids of the amino acid sequence of SEQ IDNO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984. Ina preferred embodiment, the protein has the amino acid sequence of SEQID NO:90, SEQ ID NO:102, SEQ ID NO:105, or SEQ ID NO:113.

Another embodiment of the invention features an isolated protein,preferably a SPOIL protein, having an amino acid sequence at least about60% identical to the amino acid sequence of SEQ ID NO:90, SEQ ID NO:102,SEQ ID NO:105, or SEQ ID NO:113, the amino acid sequence encoded by theDNA insert of the plasmid deposited with ATCC as Accession Number 98883,or the amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98984. Another embodiment of theinvention features an isolated protein, preferably a SPOIL protein,having an amino acid sequence at least about 85% identical to the aminoacid sequence of SEQ ID NO:102 or the amino acid sequence encoded by theDNA insert of the plasmid deposited with ATCC as Accession Number 98984.Yet another embodiment of the invention features isolated proteins,preferably SPOIL proteins, which are encoded by nucleic acid moleculeshaving a nucleotide sequence at least about 60% identical to anucleotide sequence of SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:101, SEQ IDNO:103; SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:112, SEQ ID NO:114, theDNA insert of the plasmid deposited with ATCC as Accession Number 98883,or the DNA insert of the plasmid deposited with ATCC as Accession Number98984, or a complement thereof.

This invention further features isolated proteins, preferably SPOILproteins, which are encoded by nucleic acid molecules having anucleotide sequence which hybridizes under stringent hybridizationconditions to the complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:101, SEQ IDNO:103; SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:112, or SEQ ID NO:114.

The proteins of the present invention, preferably SPOIL proteins, orportions thereof (e.g., biologically active portions thereof), can beoperatively linked to a non-SPOIL polypeptide to form fusion proteins,preferably SPOIL fusion proteins. The invention further featuresantibodies that specifically bind SPOIL proteins, such as monoclonal orpolyclonal antibodies. In addition, the proteins of the invention orbiologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of SPOIL activity or expression in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of SPOIL activity such that the presence of SPOIL activity isdetected in the biological sample.

In another aspect, the invention provides a method for modulating SPOILactivity comprising contacting a cell capable of expressing SPOIL withan agent that modulates SPOIL activity such that SPOIL activity in thecell is modulated. In one embodiment, the agent inhibits SPOIL activity.In another embodiment, the agent stimulates SPOIL activity. In oneembodiment, the agent is an antibody that specifically binds to SPOILprotein. In another embodiment, the agent modulates expression of SPOILby modulating transcription of a SPOIL gene or translation of a SPOILmRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand ofthe SPOIL mRNA or the SPOIL gene.

In another aspect, the invention provides a method for modulating IL-1activity comprising contacting a cell capable of expressing and/orresponding to IL-1 with an agent that modulates SPOIL activity such thatIL-1 activity in the cell is modulated. In one embodiment, an agentinhibits or reduces IL-1 activity. Thus, in one embodiment, the SPOILagent is a protein of the invention, preferably a SPOIL protein or abiologically active portion thereof which functions as an IL-1 receptorantagonist. In another embodiment, a SPOIL agent stimulates IL-1activity. Thus, in another embodiment, the SPOIL agent is a protein ofthe invention, preferably a SPOIL protein, SPOIL variant, orbiologically active portion thereof which functions as an IL-1 receptoragonist.

In another embodiment, the SPOIL agent is a protein of the invention,preferably a SPOIL protein or a biologically active portion thereof,which modulates an anti-inflammatory cytokine (e.g., solubleTNF-Receptor p55 (sTNFRp55), sTNFRp75 and IL-1 receptor antagonist(IL-1Ra)). In yet another embodiment, the SPOIL agent is a protein ofthe invention, preferably a SPOIL protein, SPOIL variant, orbiologically active portion thereof, which modulates a pro-inflammatorycytokine (e.g., tumor necrosis factor (TNF-α), interleukin-6 (IL-6) andinterleukin-1b (IL-1b)).

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant SPOIL and/orIL-1 expression by administering an agent which is a SPOIL modulator tothe subject. In one embodiment, the SPOIL agent is a SPOIL protein orSPOIL variant. In yet another embodiment, the SPOIL agent is a peptide,peptidomimetic, or other small molecule. In a preferred embodiment, thedisorder characterized by aberrant SPOIL and/or IL-1 expression is abone metabolism disorder, a proinflammatory disorder, or an immunedisorder.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aSPOIL protein; (ii) mis-regulation of said gene; and (iii) aberrantpost-translational modification of a SPOIL protein, wherein a wild-typeform of said gene encodes an protein with a SPOIL activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a SPOIL protein, byproviding an indicator composition comprising an a SPOIL protein havingSPOIL activity, contacting the indicator composition with a testcompound, and determining the effect of the test compound on SPOILactivity in the indicator composition to identify a compound thatmodulates the activity of a SPOIL protein.

NEOKINE

The present invention is based, at least in part, on the discovery ofnucleic acid molecules which encode a novel family of secreted proteins,referred to herein as the Neokine family of proteins (“NEOKINES” or“NEOKINE proteins”) which are ligands for a previously-identifiedputative G protein-coupled receptor termed “RDC1” also referred toherein as the “NEOKINE receptor”. The NEOKINE molecules of the presentinvention are useful as modulating agents in regulating a variety ofcellular processes. Accordingly, in one aspect, this invention providesisolated nucleic acid molecules encoding NEOKINE proteins orbiologically active portions thereof, as well as nucleic acid fragmentssuitable as primers or hybridization probes for the detection ofNEOKINE-encoding nucleic acids.

In one embodiment, a NEOKINE nucleic acid molecule is 60% homologous tothe nucleotide sequence shown in SEQ ID NO:115, SEQ ID NO:117, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98751, or complement thereof. In another embodiment,a NEOKINE nucleic acid molecule is 60% homologous to the nucleotidesequence shown in SEQ ID NO:118, SEQ ID NO:120, or a complement thereof.In yet another embodiment, a NEOKINE nucleic acid molecule is 60%homologous to the nucleotide sequence shown in SEQ ID NO:121 or SEQ IDNO:123. In yet another embodiment, a NEOKINE nucleic acid molecule is60% homologous to the nucleotide sequence shown in SEQ ID NO:124. In apreferred embodiment, an isolated NEOKINE nucleic acid molecule has thenucleotide sequence shown SEQ ID NO:117, or a complement thereof. Inanother embodiment, a NEOKINE nucleic acid molecule further comprisesnucleotides 1-96 of SEQ ID NO:115. In another embodiment, a NEOKINEnucleic acid molecule further comprises nucleotides 394-1564 of SEQ IDNO:115. In another preferred embodiment, an isolated NEOKINE nucleicacid molecule has the nucleotide sequence shown in SEQ ID NO:115.

In another preferred embodiment, an isolated NEOKINE nucleic acidmolecule has the nucleotide sequence shown SEQ ID NO:120, or acomplement thereof. In another embodiment, a NEOKINE nucleic acidmolecule further comprises nucleotides 1-211 of SEQ ID NO:118. Inanother embodiment, a NEOKINE nucleic acid molecule further comprisesnucleotides 509-1656 of SEQ ID NO:118. In another preferred embodiment,an isolated NEOKINE nucleic acid molecule has the nucleotide sequenceshown in SEQ ID NO:118.

In another preferred embodiment, an isolated NEOKINE nucleic acidmolecule has the nucleotide sequence shown SEQ ID NO:123, or acomplement thereof. In another embodiment, a NEOKINE nucleic acidmolecule further comprises nucleotides 235-1372 of SEQ ID NO:121. Inanother preferred embodiment, an isolated NEOKINE nucleic acid moleculehas the nucleotide sequence shown in SEQ ID NO:121.

In another preferred embodiment, an isolated NEOKINE nucleic acidmolecule has the nucleotide sequence shown SEQ ID NO:136, or acomplement thereof. In another embodiment, a NEOKINE nucleic acidmolecule further comprises nucleotides 285-1458 of SEQ ID NO:124. Inanother preferred embodiment, an isolated NEOKINE nucleic acid moleculehas the nucleotide sequence shown in SEQ ID NO:124.

In another preferred embodiment, an isolated NEOKINE nucleic acidmolecule is of human origin and has the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98751, ora complement thereof. In another embodiment, an isolated NEOKINE nucleicacid molecule is of rat origin. In another embodiment, an isolatedNEOKINE nucleic acid molecule is of macaque origin.

In another embodiment, a NEOKINE nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently homologous to the amino acid sequence of SEQ ID NO:116, SEQID NO:119, SEQ ID NO:122, or SEQ ID NO:125. In another preferredembodiment, a NEOKINE nucleic acid molecule includes a nucleotidesequence encoding a protein having an amino acid sequence at least 60%homologous to the amino acid sequence of SEQ ID NO:116. In yet anotherpreferred embodiment, a NEOKINE nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequence atleast 60% homologous to the amino acid sequence of SEQ ID NO:119. In yetanother preferred embodiment, a NEOKINE nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequence atleast 60% homologous to the amino acid sequence of SEQ ID NO:122. In yetanother preferred embodiment, a NEOKINE nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequence atleast 60% homologous to the amino acid sequence of SEQ ID NO:125.

In another embodiment, an isolated nucleic acid molecule of the presentinvention encodes a NEOKINE protein which includes a NEOKINE CXCsignature motif. In another embodiment, an isolated nucleic acidmolecule of the present invention encodes a NEOKINE protein whichincludes a NEOKINE CXC signature motif and a signal sequence and issecreted. In yet another embodiment, a NEOKINE nucleic acid moleculeencodes a NEOKINE protein and is a naturally occurring nucleotidesequence.

Another embodiment of the invention features NEOKINE nucleic acidmolecules which specifically detect NEOKINE nucleic acid moleculesrelative to nucleic acid molecules encoding non-NEOKINE proteins. Forexample, in one embodiment, a NEOKINE nucleic acid molecule is at least650 nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising the nucleotide sequence shown in SEQ IDNO:115, SEQ ID NO:118, SEQ ID NO:121, or SEQ ID NO:124, or a complementthereof.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a NEOKINE nucleicacid.

Another aspect of the invention provides a vector comprising a NEOKINEnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. The inventionalso provides a method for producing a NEOKINE protein by culturing in asuitable medium, a host cell of the invention containing a recombinantexpression vector such that a NEOKINE protein is produced.

Another aspect of this invention features isolated or recombinantNEOKINE proteins and polypeptides. In one embodiment, an isolatedNEOKINE protein includes a NEOKINE CXC signature motif and is secreted.In another embodiment, an isolated NEOKINE protein includes a NEOKINECXC signature motif and a signal sequence, and is secreted. In anotherembodiment, an isolated NEOKINE protein has an amino acid sequencesufficiently homologous to the amino acid sequence of SEQ ID NO:116, SEQID NO:119, SEQ ID NO:122, or SEQ ID NO:125. In a preferred embodiment, aNEOKINE protein has an amino acid sequence at least about 60% homologousto the amino acid sequence of SEQ ID NO:116. In another preferredembodiment, a NEOKINE protein has an amino acid sequence at least about60% homologous to the amino acid sequence of SEQ ID NO:119. In anotherpreferred embodiment, a NEOKINE protein has an amino acid sequence atleast about 60% homologous to the amino acid sequence of SEQ ID NO:122.In another preferred embodiment, a NEOKINE protein has an amino acidsequence at least about 60% homologous to the amino acid sequence of SEQID NO:125. In another embodiment, a NEOKINE protein has the amino acidsequence of SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ IDNO:125.

Another embodiment of the invention features an isolated NEOKINE proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequenceat least about 60% homologous to a nucleotide sequence of SEQ ID NO:115,or a complement thereof. Another embodiment of the invention features anisolated NEOKINE protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 60% homologous to anucleotide sequence of SEQ ID NO:118, or a complement thereof. Anotherembodiment of the invention features an isolated NEOKINE protein whichis encoded by a nucleic acid molecule having a nucleotide sequence atleast about 60% homologous to a nucleotide sequence of SEQ ID NO:121, ora complement thereof. Another embodiment of the invention features anisolated NEOKINE protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 60% homologous to anucleotide sequence of SEQ ID NO:124, or a complement thereof. Thisinvention further features an isolated NEOKINE protein which is encodedby a nucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:115, SEQ ID NO:118, SEQID NO:121, SEQ ID NO:124, or a complement thereof.

The NEOKINE proteins of the present invention, or biologically activeportions thereof, can be operatively linked to a non-NEOKINE polypeptideto form NEOKINE fusion proteins. The invention further featuresantibodies that specifically bind NEOKINE proteins, such as monoclonalor polyclonal antibodies. In addition, the NEOKINE proteins orbiologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

In another aspect, the present invention provides a method for detectingNEOKINE expression in a biological sample by contacting the biologicalsample with an agent capable of detecting a NEOKINE nucleic acidmolecule, protein or polypeptide such that the presence of a NEOKINEnucleic acid molecule, protein or polypeptide is detected in thebiological sample.

In another aspect, the present invention provides a method for detectingthe presence of NEOKINE activity in a biological sample by contactingthe biological sample with an agent capable of detecting an indicator ofNEOKINE activity such that the presence of NEOKINE activity is detectedin the biological sample.

In another aspect, the invention provides a method for modulatingNEOKINE activity comprising contacting the cell with an agent thatmodulates NEOKINE activity such that NEOKINE activity in the cell ismodulated. In one embodiment, the agent inhibits NEOKINE activity. Inanother embodiment, the agent stimulates NEOKINE activity. In oneembodiment, the agent is an antibody that specifically binds to aNEOKINE protein. In another embodiment, the agent modulates expressionof NEOKINE by modulating transcription of a NEOKINE gene or translationof a NEOKINE mRNA. In yet another embodiment, the agent is a nucleicacid molecule having a nucleotide sequence that is antisense to thecoding strand of a NEOKINE mRNA or a NEOKINE gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant NEOKINEprotein or nucleic acid expression or activity by administering an agentwhich is a NEOKINE modulator to the subject. In one embodiment, theNEOKINE modulator is a NEOKINE protein. In another embodiment theNEOKINE modulator is a NEOKINE nucleic acid molecule. In yet anotherembodiment, the NEOKINE modulator is a peptide, peptidomimetic, or othersmall molecule. In a preferred embodiment, the disorder characterized byaberrant NEOKINE protein or nucleic acid expression is a developmental,differentiative, proliferative disorder, an immunological disorder, orcell death.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aNEOKINE protein; (ii) mis-regulation of said gene; and (iii) aberrantpost-translational modification of a NEOKINE protein, wherein awild-type form of said gene encodes an protein with a NEOKINE activity.

The present invention also provides methods for identifying compoundswhich modulate binding of NEOKINE to the NEOKINE receptor and methodsfor identifying compounds which modulate the activity of the NEOKINEreceptor (e.g., cell-based as well as in vitro screening assays).

TANGO 129

The present invention is based, at least in part, on the discovery of agene encoding T129, a transmembrane protein that is predicted to be amember of the TNF receptor superfamily. The T129 cDNA described below(SEQ ID NO:137) has a 1290 nucleotide open reading frame (nucleotides99-1388 of SEQ ID NO:137; SEQ ID NO:139) which encodes a 430 amino acidprotein (SEQ ID NO:138). This protein includes a predicted signalsequence of about 22 amino acids (from amino acid 1 to about amino acid22 of SEQ ID NO:138) and a predicted mature protein of about 408 aminoacids (from about amino acid 23 to amino acid 430 of SEQ ID NO:138; SEQID NO:140). T129 protein possesses a Tumor Necrosis FactorReceptor/Nerve Growth Factor Receptor (“TNFR/NGFR”) cysteine-rich regiondomain (amino acids 51-90; SEQ ID NO:142). T129 is predicted to have onetransmembrane domain (TM) which extends from about amino acid 163(extracellular end) to about amino acid 186 (cytoplasmic end) of SEQ IDNO:138.

The T129 molecules of the present invention are useful as modulatingagents in regulating a variety of cellular processes. Accordingly, inone aspect, this invention provides isolated nucleic acid moleculesencoding T129 proteins or biologically active portions thereof, as wellas nucleic acid fragments suitable as primers or hybridization probesfor the detection of T129-encoding nucleic acids.

The invention features a nucleic acid molecule which is at least 45% (or55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequenceshown in SEQ ID NO:137, or SEQ ID NO:139, or a complement thereof. Theinvention features a nucleic acid molecule which includes a fragment ofat least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, or 1290) nucleotides of the nucleotide sequence shown inSEQ ID NO:137, or SEQ ID NO:139, or a complement thereof.

The invention also features a nucleic acid molecule which includes anucleotide sequence encoding a protein having an amino acid sequencethat is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical tothe amino acid sequence of SEQ ID NO:138, SEQ ID NO:140. In a preferredembodiment, a T129 nucleic acid molecule has the nucleotide sequenceshown SEQ ID NO:137, or SEQ ID NO:139.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ IDNO:138 or SEQ ID NO:140, the fragment including at least 15 (25, 30, 50,100, 150, 300, or 400) contiguous amino acids of SEQ ID NO:138 or SEQ IDNO:140.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:138 or SEQ ID NO:140, wherein the nucleic acidmolecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:137or SEQ ID NO:139 under stringent conditions.

Also within the invention are: an isolated T129 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the amino acid sequence of SEQ ID NO:140 (mature humanT129) or the amino acid sequence of SEQ ID NO:138 (immature human T129);and an isolated T129 protein having an amino acid sequence that is atleast about 85%, 95%, or 98% identical to the TNFR/NGFR cysteine-richdomain of SEQ ID NO:138 (e.g., about amino acid residues 51 to 90 of SEQID NO:138; SEQ ID NO:142).

Also within the invention are: an isolated T129 protein which is encodedby a nucleic acid molecule having a nucleotide sequence that is at leastabout 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:139; anisolated T129 protein which is encoded by a nucleic acid molecule havinga nucleotide sequence at least about 65% preferably 75%, 85%, or 95%identical the TNFR/NGFR cysteine-rich domain encoding portion of SEQ IDNO:137 (e.g., about nucleotides 248 to 368 of SEQ ID NO:137); and anisolated T129 protein which is encoded by a nucleic acid molecule havinga nucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:139.

Also within the invention is a polypeptide which is a naturallyoccurring allelic variant of a polypeptide that includes the amino acidsequence of SEQ ID NO:138 or SEQ ID NO:140, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:137 or SEQ ID NO:139 under stringentconditions.

Another embodiment of the invention features T129 nucleic acid moleculeswhich specifically detect T129 nucleic acid molecules relative tonucleic acid molecules encoding other members of the TNF receptorsuperfamily. For example, in one embodiment, a T129 nucleic acidmolecule hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:137, SEQ IDNO:139, or a complement thereof. In another embodiment, the T129 nucleicacid molecule is at least 300 (325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length andhybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence shown in SEQ ID NO:137, SEQ IDNO:139, or a complement thereof. In a preferred embodiment, an isolatedT129 nucleic acid molecule comprises nucleotides 248 to 368 of SEQ IDNO:137, encoding the TNFR/NGFR cysteine-rich domain of T129, or acomplement thereof. In another embodiment, the invention provides anisolated nucleic acid molecule which is antisense to the coding strandof a T129 nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a T129 nucleic acid molecule of theinvention. In another embodiment the invention provides a host cellcontaining such a vector. The invention also provides a method forproducing T129 protein by culturing, in a suitable medium, a host cellof the invention containing a recombinant expression vector such that aT129 protein is produced.

Another aspect of this invention features isolated or recombinant T129proteins and polypeptides. Preferred T129 proteins and polypeptidespossess at least one biological activity possessed by naturallyoccurring human T129, e.g., (1) the ability to form protein:proteininteractions with proteins in the T129 signalling pathway; (2) theability to bind T129 ligand; (3) the ability to bind to an intracellulartarget. Other activities include: (1) modulation of cellularproliferation and (2) modulation of cellular differentiation. In oneembodiment, an isolated T129 protein has a TNFR/NGFR cysteine-richdomain and lacks both a transmembrane and a cytoplasmic domain. Inanother embodiment the T129 polypeptide lacks both a transmembranedomain and a cytoplasmic domain and is soluble under physiologicalconditions.

The T129 proteins of the present invention, or biologically activeportions thereof, can be operatively linked to a non-T129 polypeptide(e.g., heterologous amino acid sequences) to form T129 fusion proteins.The invention further features antibodies that specifically bind T129proteins, such as monoclonal or polyclonal antibodies. In addition, theT129 proteins or biologically active portions thereof can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of T129 activity or expression in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of T129 activity such that the presence of T129 activity isdetected in the biological sample.

In another aspect, the invention provides a method for modulating T129activity comprising contacting a cell with an agent that modulates(inhibits or stimulates) T129 activity or expression such that T129activity or expression in the cell is modulated. In one embodiment, theagent is an antibody that specifically binds to T129 protein. In anotherembodiment, the agent modulates expression of T129 by modulatingtranscription of a T129 gene, splicing of a T129 mRNA, or translation ofa T129 mRNA. In yet another embodiment, the agent is a nucleic acidmolecule having a nucleotide sequence that is antisense to the codingstrand of the T129 mRNA or the T129 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant T129 proteinor nucleic acid expression or activity by administering an agent whichis a T129 modulator to the subject. In one embodiment, the T129modulator is a T129 protein. In another embodiment the T129 modulator isa T129 nucleic acid molecule. In other embodiments, the T129 modulatoris a peptide, peptidomimetic, or other small molecule. In a preferredembodiment, the disorder characterized by aberrant T129 protein ornucleic acid expression is a proliferative or differentiative disorder,particularly of the immune system. The present invention also provides adiagnostic assay for identifying the presence or absence of a geneticlesion or mutation characterized by at least one of: (i) aberrantmodification or mutation of a gene encoding a T129 protein; (ii)mis-regulation of a gene encoding a T129 protein; and (iii) aberrantpost-translational modification of a T129 protein, wherein a wild-typeform of the gene encodes a protein with a T129 activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a T129 protein. Ingeneral, such methods entail measuring a biological activity of a T129protein in the presence and absence of a test compound and identifyingthose compounds which alter the activity of the T129 protein.

The invention also features methods for identifying a compound whichmodulates the expression of T129 by measuring the expression of T129 inthe presence and absence of a compound.

A259 (Integrin Alpha Subunit)

This invention provides novel human and murine nucleic acid moleculeswhich encode proteins referred to herein as A259 proteins, which arenovel integrin α subunits. These proteins, fragments, derivatives, andvariants thereof are collectively referred to herein as “polypeptides ofthe invention” or “proteins of the invention”. Nucleic acid moleculesencoding the polypeptides or proteins of the invention are collectivelyreferred to as “nucleic acids of the invention”.

The A259 proteins are homologous to the α subunits of the integrinfamily, and in particular, to the α10 and α1 subunits of the integrinfamily.

The nucleic acids and polypeptides of the present invention are usefulas modulating agents in regulating a variety of cellular processes.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding a polypeptide of the invention or a biologicallyactive portion thereof. The present invention also provides nucleic acidmolecules which are suitable for use as primers or hybridization probesfor the detection of nucleic acids encoding a polypeptide of theinvention.

The invention features nucleic acid molecules which are at least 47% (or50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical tothe nucleotide sequence of SEQ ID NO:145, SEQ ID NO:163, the nucleotidesequence of the cDNA insert of a clone deposited with ATCC as AccessionNumber 207190 or 207191, or a complement thereof.

The invention features nucleic acid molecules which are at least 56% (or57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identicalto the nucleotide sequence of SEQ ID NO:146, SEQ ID NO:164, thenucleotide sequence of the cDNA insert of a clone deposited with ATCC asAccession Number 207190 or 207191, or a complement thereof.

The invention features nucleic acid molecules which include a fragmentof at least 390 (400, 500, 600, 800, 1000, 1250, 1500, 1750, 2000, 2250,2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5025,or 5042) nucleotides of the nucleotide sequence of SEQ ID NO:145, SEQ IDNO:163, the nucleotide sequence of the cDNA of ATCC Accession Number207190 or 207191, or a complement thereof.

The invention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least 44% (or 45%, 50%, 55%, 60%, 65%, 75%, 85%, 95%, or 98%)identical to the amino acid sequence of SEQ ID NO:147, SEQ ID NO:165,the amino acid sequence encoded by the cDNA of ATCC Accession Number207190 or 207191, or a complement thereof.

In preferred embodiments, the nucleic acid molecules have the nucleotidesequence of SEQ ID NO:145, 146, 163, 164, or the nucleotide sequence ofthe cDNA of ATCC Accession Number 207190 or 207191.

Also within the invention are nucleic acid molecules which encode afragment of a polypeptide having the amino acid sequence of SEQ IDNO:147, SEQ ID NO:165, or a fragment including at least 15 (25, 30, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1170, 1180, or 1188)contiguous amino acids of SEQ ID NO:147, SEQ ID NO:165, or the aminoacid sequence encoded by the cDNA of ATCC Accession Number 207190 or207191.

The invention includes nucleic acid molecules which encode a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:147, SEQ ID NO:165, or the amino acid sequenceencoded by the cDNA of ATCC Accession Number 207190 or 207191, whereinthe nucleic acid molecule hybridizes to a nucleic acid moleculeconsisting of a nucleic acid sequence encoding SEQ ID NO:147, SEQ IDNO:165, the amino acid sequence encoded by the cDNA of ATCC AccessionNumber 207190 or 207191, or a complement thereof under stringentconditions.

Also within the invention are isolated polypeptides or proteins havingan amino acid sequence that is at least about 44%, preferably 45%, 50%,55%, 60%, 65%, 75%, 85%, 95%, or 98% identical to the amino acidsequence of SEQ ID NO:147, SEQ ID NO:165, or the amino acid sequenceencoded by the cDNA of ATCC Accession Number 207190 or 207191.

Also within the invention are isolated polypeptides or proteins whichare encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 56%, preferably 57%, 58%, 59%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% identical to the nucleic acid sequenceencoding SEQ ID NO:147, SEQ ID NO:165, and isolated polypeptides orproteins which are encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:145, 146, 163, or 164, a complement thereof, or the non-codingstrand of the cDNA of ATCC Accession Number 207190 or 207191.

Also within the invention are polypeptides which are naturally occurringallelic variants of a polypeptide that includes the amino acid sequenceof SEQ ID NO:147, or the amino acid sequence encoded by the cDNA of ATCCAccession Number 207190 or 207191, wherein the polypeptide is encoded bya nucleic acid molecule which hybridizes to a nucleic acid moleculehaving the sequence of SEQ ID NO:145, 146, 163, 164, or a complementthereof under stringent conditions.

The invention also features nucleic acid molecules that hybridize understringent conditions to a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:145, 146, 163, or 164, the cDNA of ATCC AccessionNumber 207190 or 207191, or a complement thereof. In other embodiments,the nucleic acid molecules are at least 390 (400, 500, 600, 800, 1000,1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000,4250, 4500, 4750, 5000, 5025, or 5042) nucleotides in length andhybridize under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:145, 146, 163, or 164,the cDNA of ATCC Accession Number 207190 or 207191, or a complementthereof.

In one embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a nucleic acid ofthe invention.

Another aspect of the invention provides vectors, e.g., recombinantexpression vectors, comprising a nucleic acid molecule of the invention.In another embodiment, the invention provides host cells containing sucha vector or a nucleic acid molecule of the invention. The invention alsoprovides methods for producing a polypeptide of the invention byculturing, in a suitable medium, a host cell of the invention containinga recombinant expression vector such that a polypeptide is produced.

Another aspect of this invention features isolated or recombinantproteins and polypeptides of the invention. Preferred proteins andpolypeptides possess at least one biological activity possessed by thecorresponding naturally-occurring human polypeptide. An activity, abiological activity, or a functional activity of a polypeptide ornucleic acid of the invention refers to an activity exerted by aprotein, polypeptide or nucleic acid molecule of the invention on aresponsive cell as determined in vivo, or in vitro, according tostandard techniques. Such activities can be a direct activity, such asan association with or an enzymatic activity on a second protein, or anindirect activity, such as a cellular signaling activity mediated byinteraction of the protein with a second protein.

A259 biological activities include, e.g., (1) the ability to formprotein-protein interactions with proteins in the signaling pathway ofthe naturally-occurring polypeptide; (2) the ability to bind a ligand ofthe naturally-occurring polypeptide; (3) the ability to interact with anA259 ligand; and (4) the ability to modulate function, survival,morphology, migration, proliferation and/or differentiation of cells,e.g., of tissues in which it is expressed (e.g., osteoblasts, bonemarrow, neural tissue).

A259 biological activities also include, e.g., (1) the ability tomodulate, e.g., stabilize, protein-protein interactions (e.g.,homophilic and/or heterophilic), and protein-ligand interactions, e.g.,in receptor-ligand recognition; (2) ability to modulate signallingpathways; (3) ability to modulate cell-cell interactions, e.g., byacting as a cell-cell adhesion molecule, or by acting as a receptor forcell-cell adhesion molecules; (4) the ability to modulate interactionsbetween cells and proteins (e.g., extracellular matrix proteins, e.g.,collagens, fibrinogens, laminins, and fibronectin), e.g., by acting as acell surface receptor; (5) the ability to interact, e.g., noncovalentlyinteract, with an integrin β subunit; and (6) the ability to exhibit anactivity of an integrin α10 or an integrin α1 subunit.

Still other A259 biological activities include, e.g., (1) the ability tomodulate, e.g., initiate, an immune response, e.g., an inflammatoryresponse; (2) the ability to modulate, e.g., initiate wound healing,e.g., by modulating platelet aggregation or by modulating fibroblastattachment to wound sites during wound contraction; and (3) the abilityto stimulate fibrogenesis.

In one embodiment, a polypeptide of the invention has an amino acidsequence sufficiently identical to an identified domain of a polypeptideof the invention. As used herein, the term “sufficiently identical”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., with asimilar side chain) amino acid residues or nucleotides to a second aminoacid or nucleotide sequence such that the first and second amino acid ornucleotide sequences have a common structural domain and/or commonfunctional activity. For example, amino acid or nucleotide sequenceswhich contain a common structural domain having about 60% identity,preferably 65% identity, more preferably 75%, 85%, 95%, 98% or moreidentity are defined herein as sufficiently identical.

In one embodiment, an A259 protein includes at least one or more of thefollowing domains: a signal sequence, an extracellular domain, a repeatdomain, an I domain, an intergrin alpha repeat domain, a transmembranedomain, and a cytoplasmic domain. In yet another embodiment, an A259protein includes an extracellular domain and one or more repeat domains,and or one or more integrin alpha repeat domains, and is a solubleprotein. In still another embodiment, an A259 protein includes anextracellular domain, one or more repeat domains, a transmembranedomain, a cytoplasmic domain, and is a receptor protein.

The polypeptides of the present invention, or biologically activeportions thereof, can be operably linked to a heterologous amino acidsequence to form fusion proteins. The invention further featuresantibodies that specifically bind a polypeptide of the invention such asmonoclonal or polyclonal antibodies. In addition, the polypeptides ofthe invention or biologically active portions thereof can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides methods for detectingthe presence of the activity or expression of a polypeptide of theinvention in a biological sample by contacting the biological samplewith an agent capable of detecting an indicator of activity such thatthe presence of activity is detected in the biological sample.

In another aspect, the invention provides methods for modulatingactivity of a polypeptide of the invention comprising contacting a cellwith an agent that modulates (inhibits or stimulates) the activity orexpression of a polypeptide of the invention such that activity orexpression in the cell is modulated. In one embodiment, the agent is anantibody that specifically binds to a polypeptide of the invention.

In another embodiment, the agent modulates expression of a polypeptideof the invention by modulating transcription, splicing, or translationof an mRNA encoding a polypeptide of the invention. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of an mRNA encoding apolypeptide of the invention.

The present invention also provides methods to treat a subject having adisorder characterized by aberrant activity of a polypeptide of theinvention or aberrant expression of a nucleic acid of the invention byadministering an agent which is a modulator of the activity of apolypeptide of the invention or a modulator of the expression of anucleic acid of the invention to the subject. In one embodiment, themodulator is a protein of the invention. In another embodiment, themodulator is a nucleic acid of the invention. In other embodiments, themodulator is a peptide, peptidomimetic, or other small molecule.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a polypeptide of the invention, (ii) mis-regulation of a geneencoding a polypeptide of the invention, and (iii) aberrantpost-translational modification of the invention wherein a wild-typeform of the gene encodes a protein having the activity of thepolypeptide of the invention.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a polypeptide of theinvention. In general, such methods entail measuring a biologicalactivity of the polypeptide in the presence and absence of a testcompound and identifying those compounds which alter the activity of thepolypeptide.

The invention also features methods for identifying a compound whichmodulates the expression of a polypeptide or nucleic acid of theinvention by measuring the expression of the polypeptide or nucleic acidin the presence and absence of the compound

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of sequence comparisons between portions of the aminoacid sequence of T85 and a fibronectin type III domain (PF00041) and anIg superfamily domain (PF00047) derived from a hidden Markov model.

FIGS. 2A-D depict a comparison of the amino acid sequence of T85 andhuman Robo protein.

FIG. 3 depicts an alignment of an amino acid sequence of Tango-77 (T77;SEQ ID NO:2) with IL4RA (SEQ ID NO:14), and IL-1β (SEQ ID NO:15).

FIG. 4 depicts an alignment of the amino acid sequence of murine SPOIL-I(also referred to as murine or mTANGO 080-1) (corresponding amino acids1 to 98 of SEQ ID NO:2), murine IL-1ra (Swissprot™ Accession NumberP25085) (SEQ ID NO:10), murine IL-1α (Swissprot™ Accession NumberP01582) (SEQ ID NO:11) and murine IL-1β (Swissprot™ Accession NumberP10749) (SEQ ID NO:12).

FIGS. 5A-D depict pairwise alignments of SPOIL amino acid sequences ofthe present invention. FIG. 5A depicts an alignment of human SPOIL-Iwith human SPOIL-II. FIG. 5B depicts an alignment of murine SPOIL-I withmurine SPOIL-II. FIG. 5C depicts an alignment human SPOIL-I with murineSPOIL-I. FIG. 5D depicts an alignment of human SPOIL-II with murineSPOIL-II. The alignments were generated using the ALIGN algorithm (Myersand Miller (1989) CABIOS). Gap penalties were set at −12/−4 and a PAM120residue weight matrix was used.

FIG. 6 depicts a multiple sequence alignment of the amino acid sequenceof murine SPOIL-I (corresponding to SEQ ID NO:2), the amino acidsequence of murine SPOIL-II (corresponding to SEQ ID NO:25), the aminoacid sequence of human SPOIL-I (corresponding to SEQ ID NO:14), and theamino acid sequence of human SPOIL-II (corresponding to SEQ ID NO:18).Asterisks indicate amino acid residues that are conserved between SPOILfamily members.

FIG. 7 depicts a multiple sequence alignment of the amino acid sequenceof murine IL-1α (Swissprot™ Accession Number P01582) (SEQ ID NO:11),murine IL-1β (Swissprot™ Accession Number P10749) (SEQ ID NO:12), murineIL-1ra (Swissprot™ Accession Number P25085) (SEQ ID NO:10), the aminoacid sequence of murine SPOIL-I (also referred to as murine or mTANGO080-I) (corresponding amino acids 1 to 98 of SEQ ID NO:2), the aminoacid sequence of murine SPOIL-II (also referred to as murine or mTANGO080-II (corresponding amino acids 1 to 160 of SEQ ID NO:25), the aminoacid sequence of human SPOIL-I (corresponding nucleotides 1 to 169 ofSEQ ID NO:14), and the amino acid sequence of human SPOIL-II(corresponding to nucleotides 1 to 208 of SEQ ID NO:16). Asterisksindicate amino acid residues that are conserved between SPOIL proteinsand IL-1ra.

FIG. 8 is a diagram depicting the relationship between the NEOKINEproteins of the instant invention. The figure depicts the functionaldomains of the NEOKINE family members, human NEOKINE-1 (SEQ ID NO:2),mouse NEOKINE-1 (SEQ ID NO:5), rat NEOKINE-1 (SEQ ID NO:8), and macaqueNEOKINE-1 (SEQ ID NO:21). The NEOKINE CXC signature motifs are indicatedin italics. The conserved cysteine residues are indicated withasterisks.

FIG. 9 is a hydropathy plot of T129. The location of the predictedtransmembrane (TM), cytoplasmic (IN), and extracellular (OUT) domainsare indicated as are the position of cysteines (cys; vertical barsimmediately below the plot). Relative hydrophilicity is shown above thedotted line, and relative hydrophobicity is shown below the dotted line.

FIG. 10 depicts a hydropathy plot of human A259. Relatively hydrophobicregions of the protein are above the dashed horizontal line, andrelatively hydrophilic regions of the protein are below the dashedhorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence (amino acids 1 to 22 of SEQ ID NO:3; SEQ ID NO:5) on theleft from the mature protein (amino acids 23 to 1188 of SEQ ID NO:3; SEQID NO:4) on the right. Thicker gray horizontal bars below the dashedhorizontal line indicate extracellular (“out”), transmembrane (“TM”),and intracellular (“in”) regions of the molecule.

FIG. 11 depicts a hydropathy plot of murine A259. Relatively hydrophobicregions of the protein are above the dashed horizontal line, andrelatively hydrophilic regions of the protein are below the dashedhorizontal line. The cysteine residues (cys) and potentialN-glycosylation sites (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The dashed vertical line separates thesignal sequence (amino acids 1 to 22 of SEQ ID NO:21; SEQ ID NO:23) onthe left from the mature protein (amino acids 23 to 1188 of SEQ IDNO:21; SEQ ID NO:22) on the right. Thicker gray horizontal bars belowthe dashed horizontal line indicate extracellular (“out”), transmembrane(“TM”), and intracellular (“in”) regions of the molecule.

FIG. 12A depicts an alignment of amino acids 37-90 of human A259 (SEQ IDNO:35) with a consensus integrin alpha repeat domain derived from ahidden Markov model (SEQ ID NO:40).

FIG. 12B depicts an alignment of amino acids 421-472 of human A259 (SEQID NO:36) with a consensus integrin alpha repeat domain derived from ahidden Markov model (SEQ ID NO:40).

FIG. 12C depicts an alignment of amino acids 476-532 of human A259 (SEQID NO:37) with a consensus integrin alpha repeat domain derived from ahidden Markov model (SEQ ID NO:40).

FIG. 12D depicts an alignment of amino acids 538-593 of human A259 (SEQID NO:38) with a consensus integrin alpha repeat domain derived from ahidden Markov model (SEQ ID NO:40).

FIG. 12E depicts an alignment of amino acids 600-654 of human A259 (SEQID NO:39) with a consensus integrin alpha repeat domain derived from ahidden Markov model (SEQ ID NO:40).

FIG. 13A is a graph depicting the results of an analysis of A259expression in three normal liver clinical samples (A, B, and C) andeight liver fibrosis clinical samples (D, E, F, G, H, I, J, and K).

FIG. 13B is a graph depicting the results an analysis of human A259expression in a variety of cells: heart (A), lung (B), liver (C),passaged stellate cells (D), quiescent stellate cells (E), srellatecells (F) stellate/F BS cells (G), NHDF fibroblasts (H); TGF-treatedNHDF fibroblasts (I); NHLF fibroblasts (J); and TGF-treated NHLFfibroblasts (K).

DETAILED DESCRIPTION OF THE INVENTION Delta 3

Notch, first identified in Drosophila, is the founding member of afamily of transmembrane receptor proteins that mediate cell responses tointrinsic and/or extrinsic developmental cues. The cellular response toNotch signaling can be differentiation, proliferation and/or apoptosisdepending on the specific developmental program. Defects in the Notchsignaling pathway may be involved in neurological, vascular andhematologic diseases.

Analysis of gene expression patterns for Notch and its ligands hasindicated that Notch signaling may have a role in hematopoiesis (Milnerand Bigas (1999) Blood 93:2431). Notch-1 was also shown to be involvedin determination of T-cell education and fate in the thymus (Robey(1999) An Rev Immunol 17:283). Furthermore, a subset of human T-cellleukemia patients harbor a translocation involving the Notch 1 genewhich results in a constitutively active Notch protein (Ellisen et al.(1991) Cell 66:649). Compelling evidence that the Notch signalingpathway is involved in B-cell development is seen in B-cell malignanciesinduced by Epstein-Barr virus (EBV). EBNA2, the transforming protein ofEBV, transactivates cellular genes by direct interaction with a primarycomponent of the Notch pathway (Henkel et al. (1994) Science 265:92).

It has recently been shown that the human Notch3 gene, located onchromosome 19, is mutated in CADASIL patients (Joutel et al., (1996)Nature 383: 707-710). CADASIL causes a type of stroke and dementia whosekey features include recurrent subcortical ischemic events, progressivevascular dementia, craniofacial paralysis, migraine and mood disorderswith severe depression (Chabriat et al., (1995) Lancet 346: 934-939).Pathological examination reveals multiple small, deep cerebral infarcts,a leukoencephalopathy and a non-atherosclerotic, non-amyloid angiopathyinvolving mainly the small cerebral arteries (Baudrimont et al., (1993)Stroke 24: 122-125). Severe alterations of vascular smooth muscle cellsare evident on ultrastructural analysis (Ruchoux et al., (1995) Acta.Neuropathol. 89:500-512). Therefore, disruption of the Notch signalingpathway appears to be responsible for CADASIL stroke and dementia.Defects in the Notch signaling pathway may also be involved in otherneurological diseases, e.g., Alzheimer's disease.

The Notch signaling pathway comprises Notch proteins, which are membraneproteins, and proteins interacting with Notch proteins, termed Deltaproteins. The product of the Delta gene, acting as a ligand, and that ofthe Notch gene, acting as a receptor, are key components in alateral-inhibition signaling pathway that regulates the detailedpatterning of many different tissues in Drosophila (Vassin et al.,(1987) EMBO J. 6:3431-3440; Kopczynski et al., (1988) Genes Dev.2:1723-1735; Fehon et al., (1990) Cell 61:523-534; Artavanis-Tsakonas etal., (1991) Trends, Genet. Sci. 7:403-407; Heitzler et al., (1991) Cell64: 1083-1092; Greenwald et al., (1992) Cell 68: 271-281; Fortini etal., (1993) Cell 75: 124501247; and Muskavitch (1994) Devl. Biol.166:415-430). During neurogenesis in particular, neural precursors, byexpressing Delta, inhibit neighboring Notch-expressing cells frombecoming committed to a neural fate. Mutations leading to a failure oflateral inhibition cause an overproduction of neurons, giving rise to aphenotype termed the “neurogenic phenotype” in Drosophila. For example,loss of Notch 1 leads to somite defects and embryonic death in mice,whereas constitutively active mutant forms of Notch 1 appear to inhibitcell differentiation in Xenopus and in cultured mammalian cells (Swiateket al. (1994) Genes Dev. 8:707; Conlon et al. (1995) J. Development121:1533; Lopan et al. (1994) Development 120:2385; and Nye et al.(1994) Development 120:2421). Furthermore, loss of Dll1 function in miceleads to excessive neuronal differentiation, resulting in severepatterning defects in the paraxial mesoderm and a hyperplastic centralnervous system (CNS) (Hrabe de Angelis et al. (1997) Nature 386:717).Thus, the Notch signaling pathway, in particular Delta proteins, mediatelateral inhibition during neurogenesis so that only a limited proportionof cells having the potential to become neurons will in factdifferentiate into neurons.

The Notch family of proteins are transmembrane receptors containingseveral conserved peptide motifs. The extracellular domains contain manytandemly repeated copies of an epidermal growth factor (EGF) like motif.The intracellular domains contain six copies of another conserved motif,termed the Cdc10/ankyrin repeat.

A protein interacting with Notch was first discovered in Drosophila andhas been called Delta protein. This protein encodes a transmembraneprotein ligand, which contains tandem arrays of epidermal growthfactor-like repeats in the extracellular domain. The Delta and Notchproteins can directly bind to each other and specific EGF-like domainsare sufficient and necessary for this binding (Fehon et al., (1990) Cell61:523-534; Rebay et al., (1991) Cell 67:687-699; and Lieber et al.,(1992) Neuron 9: 847-859).

It is also possible that soluble forms of the protein also exist. Suchsoluble isoforms can arise through variable splicing of the Delta3 geneor alternatively as a result of proteolysis of a membranous isoform. Infact, a splice variant of a chicken Delta protein have been described inPCT Publication No. WO 97/01571 (Jan. 16, 1997). Furthermore, the humanDelta-like polypeptide Dlk is a soluble protein (Jansen et al. (1994)Eur. J. Biochem. 225:83-92).

In addition to the Drosophila Delta protein, a chick Delta ortholog,C-Delta protein (Henrique et al., (1995) Nature 375: 787-790 and GenBankAccession No. U26590) two Xenopus orthologs, X-Delta-1 and X-Delta-2(Chitnis et al., (1995) Nature 375:761-766 and GenBank Accession Nos.L42229 and U70843), a mouse ortholog (GenBank Accession No. X80903), adelta-like human gene 1(Dlk) (Bettenhausen et al., (1995) Development121:2407-2418) a rat ortholog (GenBank Accession No. U78889), and aZebrafish ortholog (GenBank Accession No. Y11760) have been identified.

The present invention is based at least in part on the discovery of anovel gene encoding a human Delta protein referred to herein as“hDelta3” polypeptide, and the mouse equivalent referred to herein as“mDelta3”. An exemplary hDelta3 has been deposited with the ATCC® onMar. 5, 1997 and has been assigned ATCC® GenBank Accession Number 98348.The human Delta3 gene maps to human chromosome 15.

The DNA sequence of human Delta3 including 5′ and 3′ non-codingsequences is shown in SEQ ID NO:1, the coding sequence is shown in SEQID NO:3, and the deduced amino acid sequence of the human Delta3 proteinis shown in SEQ ID NO: 2. The DNA sequence of mouse Delta3 including 5′and 3′ non-coding sequences is shown in SEQ ID NO:24 and the codingsequence is shown in SEQ ID NO:26. The deduced amino acid sequence ofthe mouse Delta3 protein is shown in SEQ ID NO:25.

Human Delta3 is expressed in endothelial cells and in fact was clonedfrom a human microvascular endothelial cell library. Northern blotanalysis of RNA prepared from a number of different human tissues,indicate that a 3.5 kb Delta3 mRNA transcript is present in fetal brain,lung, liver and kidney, and adult heart, placenta, lung, skeletalmuscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, smallintestine and colon. Low levels of Delta3 mRNA were also detected inadult brain and adult liver. However, no Delta3 mRNA was detected inperipheral blood leukocytes. These results indicate that Delta3 isexpressed in a tissue-specific manner. Further, expression in humanmicrovascular endothelial cells was found to be up-regulated (about 2-3fold) in cells that had been stimulated with certain growth factors(e.g., basic fibroblast growth factor (bFGF) or vascular endothelialgrowth factor (VEGF)). In addition, strong expression of human Delta3was observed in the colorectal carcinoma cell line, SW480. Furthermore,expression of hDelta3 has been shown to be induced in response toproliferation and differentiation signals (See Examples). Thus, theDelta3 gene, in particular, the hDelta3 gene, is a gene whose expressionin a cell changes with the state of proliferative and/or differentiativestate of cells.

In situ hybridization was performed on a wide range of murine adult andembryonic tissues using a probe complementary to mRNA of mDelta3.Expression was most abundant and widespread during embryogenesis.Strongest expression was observed in the eye of all the embryonic agestested. Signal in a pattern suggestive of neuronal expression was notobserved in any other tissues making the expression in the eye unique.Ubiquitous expression was also detected in lung, thymus and brown fatduring embryogenesis. A multifocal, scattered signal was also observedthroughout the embryo. This signal pattern was more focused in thecortical region of the kidney and outlining the intestinal tract. Adultexpression was highest in the ovary and the cortical regions of thekidney and adrenal gland. This is consistent with Delta3's role as aregulator of cell growth and/or differentiation.

As predicted from the nucleotide sequence of the nucleic acid encodinghDelta3, the novel, full-length hDelta3 polypeptide comprises 685 aminoacids and is similar and structure to Delta proteins obtained from otherorganisms (discussed below). An amino acid sequence analysis of Delta3proteins predicts that the protein comprises at least the structuraldomains described herein. First, human and mouse Delta3 have a signalpeptide, corresponding to amino acid 1 to amino acid 16, amino acid 1 toamino acid 17, amino acid 1 to amino acid 18, amino acid 1 to amino acid19 or amino acid 1 to amino acid 20 of SEQ ID NOs: 2 or 25. The signalsequence is normally cleaved during processing of the mature protein. Insuch embodiments of the invention, the domains and the mature proteinresulting from cleavage of such signal peptides are also includedherein. For example, the cleavage of a signal sequence consisting ofamino acids 1 to 17 results in an extracellular domain consisting ofamino acids 18 to 529 of SEQ ID No. 2 and the mature Delta3 polypeptidecorresponding to amino acids 18 to 685 of SEQ ID NO: 2.

Second, human and mouse Delta3 have protein interaction domains such asa Delta Serrated lag-2 (DSL) motif corresponding to amino acid 173 toamino acid 217 of SEQ ID NO: 2 and amino acid 174 to amino acid 218 ofSEQ ID NO: 25, as well as eight epidermal growth factor (EGF)-likerepeats.

In addition, Delta3 proteins have a transmembrane domain, i.e., in humanDelta3 the transmembrane domain corresponds to about amino acid 530 toabout amino acid 553 of SEQ ID NO: 2, and in mouse Delta3, amino acid531 to amino acid 554 of SEQ ID NO: 25. Delta3 proteins have also have acytoplasmic domain corresponding to about amino acid 554 to about aminoacid 685 of SEQ ID NO: 2 or amino acid 555 to amino acid 686 of SEQ IDNO: 25. Accordingly, sequence analysis for conserved domains of Delta3amino acid sequence shows that the protein is likely a transmembraneprotein having an extracellular domain corresponding to about amino acid1 to about amino acid 529 of SEQ ID NO:2, about amino acid 18 to aboutamino acid 529 of SEQ ID NO: 2, amino acid 1 to about amino acid 530 ofSEQ ID NO: 25, or amino acid 18 to 530 of SEQ ID NO: 25, saidextracellular domain comprising a DSL motif and eight EGF-like domains.The Delta3 protein further comprises a transmembrane domain and acytoplasmic domain.

Human Delta3 protein is similar in structure and in sequence to theDelta proteins identified in Drosophila, Xenopus, zebrafish, chicken,rat, mouse, rat, and human. Applicants have aligned known Delta proteinswith the Delta3 protein of the present invention. This alignmentcontains the following Delta proteins: a mouse Delta1 protein(m-delta1), rat Delta-1 protein (r-delta1), a human Delta-1 protein(h-delta1), a Xenopus Delta1 protein (x-delta1), a chicken Delta1protein (c-delta1), a zebrafish Delta1 protein (z-delta1), a secondXenopus Delta protein (x-delta2), as well as the human Delta3 protein(h-delta3), and a Drosophila Delta1 protein (d-delta). The amino acidsequence of h-delta1 is the amino acid sequence published in PCTPublication WO 97/01571 (Jan. 16, 1997) which is incomplete and containsnumerous errors, as stated in the application. Since the amino acidsequence alignment has been done using the pileup computer program (GCGPackage), the order of the amino acid sequences reflects the relativeidentity between the different Delta proteins. Accordingly, theDrosophila protein, which corresponds to the bottom sequence in thealignment is most distant from the other Delta proteins.

The alignment shows that hDelta3, which is listed second to the last, isthe second most distant Delta protein from the previously identifiedmouse, rat, human, Xenopus, zebrafish, and chicken delta protein.Accordingly, hDelta3 protein is significantly different from thepreviously described human Delta protein, as well as the Delta proteinsfrom the other species. Interestingly, the hDelta3 protein has an aminoacid sequence which is equally distant from both Xenopus proteins, i.e.,Delta1 and Delta2, suggesting that hDelta3 does not correspond to eitherof the Xenopus Delta proteins. Therefore, the newly isolated polypeptidehas been termed hDelta3 and the previously identified mouse, rat, human,zebrafish, and Xenopus Delta proteins are termed Delta1 proteins hereinand the two Xenopus proteins are termed Delta1 and Delta2 proteins. Thedifference between hDelta3 protein and previously isolated Deltaproteins can also be visualized by comparing the percentage similarityor identity between hDelta3 and the previously identified Delta1 andDelta2 proteins on one hand (Table I), and the percent similarity oridentity of a Delta1 protein with the other Delta1 and Delta2 proteins(Table II).

A hallmark of Notch ligands such as Jagged-1, is the ability to blockthe differentiation of the C2C12 cell line from myoblasts into myotubeswhen co-cultured with NIH3T3 cells under low mitogenic conditions. WhenC2C12 cells were co-cultured with NIH3T3 cells, which were engineered toexpress hDelta3, differentiation of C2C12 cells from myoblasts tomyotubes was blocked in a similar fashion as has been described forother Notch ligands such as Jagged-1. Therefore, the hDelta3 gene islikely to encode a polypeptide which functions as a bona fide Notchligand. Indeed, the data presented in Section 5.6, below, indicates thathDelta3 is a bona fide Notch ligand.

Table I indicates the percent similarity and identity between humanDelta3, the Delta1 disclosed in PCT Publication No. WO 97/01571 (Jan.16, 1997) and non-human Delta1 proteins. Since the amino acid sequenceof the human Delta1 protein that is disclosed in PCT Publication No. WO97/01571 (Jan. 16, 1997) is incomplete, the percentage similarity andidentity was determined using a portion of the human Delta1 amino acidsequence which seems most reliable. The portion of the amino acidsequence used corresponds to amino acids 214-370 of the human Delta1amino acid sequence shown in FIG. 11 of the PCT Publication No. WO97/01571 (Jan. 16, 1997).

TABLE I Percentage similarity between the amino acid sequence of humanDelta3 (SEQ ID NO: 2) and that of the various Delta proteins Accession %% GenBank NO: SEQ ID NO: identity similarity Human Delta1 N.A. (SEQ IDNO: 6) 50 66 Mouse Delta1 X80903 (SEQ ID NO: 4) 53 70 rat Delta1 U78889(SEQ ID NO: 5) 54 70 chicken Delta1 U26590 (SEQ ID NO: 8) 52 68 XenopusDelta1 L42229 (SEQ ID NO: 7) 51 68 zebrafish Delta1 Y11760 (SEQ ID NO:9) 48 67 Xenopus Delta2 U70843 (SEQ ID NO: 10) 47 65 Drosophila AA142228(SEQ ID NO: 11) 40 58 Delta1 hDelta-like (dlk) U15979 33 55

Table II indicates the percent similarity and identity between humanDelta1 disclosed in PCT Publication No. WO 97/01571 (1997) and non-humanDelta1 proteins. Since the amino acid sequence of the human Delta1protein that is disclosed in PCT Publication No. WO 97/01571 (1997) isincomplete, the percentage similarity and identity was determined usinga portion of the human Delta1 amino acid sequence which seems mostreliable. The portion of the amino acid sequence used corresponds toamino acids 214-370 of the human Delta1 amino acid sequence shown inFIG. 11 of the PCT application.

TABLE II Percentage similarity between human Delta1 and the variousnon-human Delta1 or Delta2 proteins % % similarity GenBank Accession SEQID NO: identity NO: Human Delta1 N.A. (SEQ ID NO: 6) 100 100 mouseDelta1X80903 (SEQ ID NO: 4) 86 95 rat Delta1 U78889 (SEQ ID NO: 5) 88 94chicken Delta1 U26590 (SEQ ID NO: 8) 85 89 Xenopus Delta1 L42229 (SEQ IDNO: 7) 78 84 zebrafish Delta1 Y11760 (SEQ ID NO: 9) 69 80 Xenopus Delta2U70843 (SEQ ID NO: 10) 57 70 Drosophila AA142228 (SEQ ID NO: 11) 45 62Delta1 hDelta-like (dlk) U15979 37 55

Accordingly, Table I indicates that hDelta3 is only approximately 66%similar to the human Delta1 protein; approximately 70% similar to themouse Delta1 protein; approximately 70% similar to the rat Delta1protein; approximately 68% similar to the chick Delta1 protein;approximately 68% similar to the Xenopus Delta1 protein, approximately70% similar to the Xenopus Delta2 protein and approximately 58% similarto the Drosophila Delta1 protein. However, as shown in Table II, thehuman-Delta1 protein is very similar to the mouse, rat, chick, Xenopus,zebrafish, and Drosophila Delta1 and the Xenopus Delta2 proteins. Inaddition, mouse and rat Delta1 proteins are about 95% similar. Thus, theamino acid sequence of the orthologs of the Delta1 protein share greatersimilarity and identity with each other than with the human Delta3protein of the invention, indicating that at least two families of Deltaproteins exist.

The difference between the newly isolated hDelta3 protein and thepreviously identified Delta1 and Delta2 proteins can also be seen bycreating a phylogenic tree using the Growtree Phylogram computer program(GCG Package). The result of this analysis, indicates that h-Delta3 ison a different “branch” in the phylogenic tree from the other Deltaproteins, thus confirming that hDelta3 protein is more distant from theother Delta1 and Delta2 proteins than they are distant from each other.According to the analysis, and as predicted by the sequence alignment,only the Drosophila Delta protein is more distantly related to thepreviously identified mouse, rat, Xenopus, chicken, zebrafish and humanDelta proteins than hDelta3. Thus, the newly isolated hDelta3 protein isa member of a different subspecies of the family of Delta proteins.

Notwithstanding that each animal species is likely to have at least twoor three members (e.g., Delta1, Delta 2, and Delta3), the DSL region,the eight EGF repeats and the TM appear to be highly conservedthroughout species. However, these domains of the hDelta3 protein differmore from the corresponding domains in the other Delta proteins than thecorresponding domains in the other Delta differ from one another.

A comparison of human and mouse Delta3 shows that they are 86.6%identical and 88.2% similar. The polypeptides were aligned with theBLAST program using Blosum62, gap weight 12 and length weight 4. One gapwas introduced at amino acid position twenty one due to an extra codonpresent in mouse Delta3. The skilled artisan will appreciate that thedomains identified in hDelta3 protein of the present invention are alsopresent in corresponding positions in mouse Delta3.

Furthermore, as set forth in the examples presented below, Delta3 hasbeen localized to human chromosome 15 in a region close to the frameworkmarkers D15S1244 and D15S144. Interestingly, the region on chromosome 15that is flanked by the markers D15S1040 and D15S118 has been shown to begenetically linked with the disease called Agenesis of the CorpusCallosum with Peripheral Neuropathy (ACCPN) (Casaubon et al. (1996) AmJ. Hum. Genet. 58:28). No specific gene has so far been linked to thisdisease. Accordingly, since Delta3 is localized to a chromosomal regiongenetically linked to ACCPN and Delta3 is a member of the Notchsignaling pathway, defects in which have been associated with a numberof neurological diseases or conditions, Delta3 is likely to be the geneinvolved in ACCPN.

ACCPN, which is also termed Andermann syndrome (MIM 218000), is anautosomal recessive disorder that occurs with a high prevalence in theFrench Canadian population in the Charlevoix and Saguenay-Lac St Jeanregion in Quebec. The disease is characterized by a progressiveperipheral neuropathy caused by axonal degeneration and a centralnervous system (CNS) malformation characterized by the absence ofhypoplasia of the corpus callosum. The disorder appears early in life,is progressive and results in death in the third decade of life of thesubject.

Accordingly, certain aspects of the present invention relate to Delta3proteins, nucleic acid molecules encoding Delta3 proteins, antibodiesimmunoreactive with Delta3 proteins, and preparations of suchcompositions. In addition, drug discovery assays are provided foridentifying agents that modulate the biological function of Deltaproteins, e.g., Delta3 proteins (i.e. agonists or antagonists), such asby binding to Delta3 or by altering the interaction of Delta3 witheither downstream or upstream elements in the Delta/Notch signaltransduction pathway by altering the interaction between Delta3 and aDelta3 binding protein. Such agents can be useful therapeutically, forexample, to alter cell growth and/or differentiation or induction ofapoptosis. Moreover, the present invention provides diagnostic andtherapeutic assays and reagents for detecting and treating disordersinvolving an aberrant Delta3 activity, for example, aberrant expression(or loss thereof) of Delta3 gene or which are associated with a specificDelta allele, e.g., a Delta3 allele.

The term “activity,” for the purposes herein refers to an activityexerted by a polypeptide of the invention on a responsive cell asdetermined, in vivo or in vitro, according to standard techniques. Anactivity can refer to an effector or antigenic function that is directlyor indirectly performed by a Delta3 polypeptide (whether in its nativeor denatured conformation), or by any subsequence thereof. Effectorfunctions include, for example, receptor binding or activation,induction of differentiation, mitogenic or growth promoting activity,induction of apoptosis, signal transduction, immune modulation, DNAregulatory functions and the like, whether presently known or inherent.Antigenic functions include possession of an epitope or antigenic sitethat is capable of binding antibodies raised against anaturally-occurring or denatured Delta3 polypeptide or fragment thereof.Accordingly, an activity of a Delta3 protein can be binding to areceptor, such as Notch. An activity of a Delta3 protein can also bemodulation of cell proliferation and/or differentiation, or cell deathin a target cell having an appropriate receptor. A target cell can be,e.g., a neural cell, an endothelial cell, or a cancer cell.

The term “aberrant Delta3 activity” or “abnormal Delta3 activity” isintended to encompass an activity of Delta3 which differs from the sameDelta3 expression or activity in a healthy subject. An aberrant Delta3activity can result, e.g., from a mutation in the protein, whichresults, e.g., in lower or higher binding affinity to a receptor. Anaberrant Delta3 activity can also result from a lower or higher level ofDelta3 on cells, which can result, e.g., from aberrant transcription,splicing, or translation of the Delta3 gene. For example, an aberrantDelta3 activity can result from an abnormal promoter activity. Anaberrant Delta3 activity can also result from an aberrant signallingthrough the cytoplasmic domain of the Delta3 protein, such that, e.g.,an aberrant signal is transduced. Aberrant signalling can result from amutation in the cytoplasmic domain of Delta3 or, alternatively, from thecontact with an abnormal cytoplasmic protein. An aberrant Delta3activity can also result from contact of a Delta3 protein with anaberrant receptor, e.g., abnormal Notch protein.

The term “allele”, which is used interchangeably herein with “allelicvariant” refers to alternative forms of a gene, nucleic acid or portionsthereof, as well as to a polypeptide encoded by said gene, nucleic acid,or portion thereof. Nucleic acid alleles occupy the same locus orposition on homologous chromosomes. When a subject has two identicalalleles of a gene, the subject is said to be homozygous for the gene orallele. When a subject has two different alleles of a gene, the subjectis said to be heterozygous for the gene. Alleles of a specific gene candiffer from each other in a single nucleotide, or several nucleotides,and can include substitutions, deletions, and insertions of nucleotides.An allele of a gene can also be a form of a gene containing a mutation.

The term “allelic variant of a polymorphic region of a Delta3 gene”refers to a region of a Delta gene having one of several nucleotidesequences found in that region of the gene in other individuals, as wellas to polypeptides encoded by nucleic acid molecules comprising saidsequences.

The term “agonist”, as used herein, is meant to refer to an agent thatupregulates (e.g., potentiates or supplements) Delta3 expression, levelsand/or activity. It is to be understood that a Delta3 agonist caninclude a compound which increases signaling from a Delta3 protein,e.g., a compound bound to Delta3, such as a stimulatory form of atoporythmic protein or a small molecule. A Delta3 agonist can also, forexample, be a compound which modulates the expression or activity of aprotein which is located upstream or downstream of Delta3 and or whichinteracts with Delta3.

“Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g., suppresses or inhibits) Delta3 expression, levelsand/or activity. A Delta3 antagonist can, for example, be a compoundwhich decreases signalling from a Delta3 protein, e.g., a compoundbinding to Delta3 such as an inhibitory form of a toporythmic protein,or a small molecule. A Delta3 antagonist can include compounds thatinhibit the interaction between a Delta3 protein and another molecule,e.g., a toporythmic protein. A Delta3 antagonist can also be a compoundwhich modulates the expression or activity of a protein which is locatedupstream or downstream of Delta3 and/or which interacts with Delta3.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen biding site which specifically bindsan antigen, such as a polypeptide of the invention, e.g., an epitope ofa polypeptide of the invention. A molecule

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically bindsan antigen, such as a polypeptide of the invention, e.g., an epitope ofa polypeptide of the invention. A molecule which specifically binds to agiven polypeptide of the invention is a molecule which binds thepolypeptide, but does not substantially bind other molecules in asample, e.g., a biological sample, which naturally contains thepolypeptide. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

“Complementary” sequences as used herein refer to sequences which havesufficient complementarity to be able to hybridize, forming a stableduplex.

A “delivery complex” shall mean a targeting means (e.g., a molecule thatresults in higher affinity binding of a gene, protein, polypeptide orpeptide to a target cell surface and/or increased cellular uptake by atarget cell). Examples of targeting means include: sterols (e.g.,cholesterol), lipids (e.g., a cationic lipid, virosome or liposome),viruses (e.g., adenovirus, adeno-associated virus, and retrovirus) ortarget cell specific binding agents (e.g., ligands recognized by targetcell specific receptors). Preferred complexes are sufficiently stable invivo to prevent significant uncoupling prior to internalization by thetarget cell. However, the complex is cleavable under appropriateconditions within the cell so that the gene, protein, polypeptide orpeptide is released in a functional form.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding a Delta3 polypeptide” may thus refer to one or moregenes within a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a protein with the same biological activity.

The term “Delta3 therapeutic” refers to various compositions of Delta3modulators (e.g., agonists or antagonists), such as polypeptides,antibodies, peptidomimetics, small molecules and nucleic acids which arecapable of mimicking or potentiating (agonizing) or inhibitingsuppressing (antagonizing) Delta3 expression, levels, or activity, e.g.,which are capable of agonizing or antagonizing the effects of anaturally-occurring Delta3 protein.

The terms “Delta3 polypeptide” and “Delta3 protein” are intended toencompass, e.g., Delta3 polypeptides which have at least one activity ofa native Delta3 polypeptide, or can, e.g., antagonize or agonize atleast one biological activity of a native Delta3 polypeptide.

A “fusion protein” is a fusion of a first amino acid sequence encodingone of the subject Delta3 polypeptides with a second, heterologous aminoacid sequence. In general, a fusion protein can be represented by thegeneral formula X-Delta3-Y, wherein Delta3 represents a portion of theprotein which is derived from one of the Delta3 proteins of theinvention, and X and Y are independently absent or represent amino acidsequences which are heterologous to (that is, not related to) one of theDelta3 sequences in an organism, including naturally-occurring mutants.Among the Delta3 fusion protein is a Delta3-Ig fusion protein.

As used herein, the term “gene” or “recombinant gene”, as applied toDelta3, refers to a nucleic acid molecule comprising an open readingframe encoding one of the Delta3 polypeptides of the present invention.In one embodiment, these terms relate to a cDNa sequence including, butnot limited to a nucelci acid sequence obtained via reversetranscription of an mRNA molecule.

The term “growth state” of a cell refers to the proliferative state of acell as well as to its differentiative state. Accordingly, the termrefers to the phase of the cell cycle in which the cell is, e.g., G0,G1, G2, prophase, metaphase, or telophase, as well as to its state ofdifferentiation, e.g., undiffereniated, partially differentiated, orfully differentiated. Without wanting to be limited, differentiation ofa cell is usually accompanied by a decrease in the proliferative rate ofa cell.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. Likewise, a degree of identity of nucleic acidsequences is a function of the number of identical nucleic acids atpositions shared by the nucleic acid sequences.

Furthermore, a degree of homology or similarity of amino acid sequencesis a function of the number of conserved amino acids at positions sharedby the amino acid sequences. A sequence which is “unrelated” or“non-homologous” with one of the hDelta3 sequences of the presentinvention typically is a sequence which shares less than 40% identity,though preferably less than 25% identity with one of the hDelta3sequences of the present invention.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength. In another embodiment, the mouse Delta3 polypeptide is one aminoacid longer than human Delta3.

Preferably, the determination of percent identity between two sequencesis accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlinand Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to a protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. Id. When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, (1988)CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75% ormore) identical to each other typically remain hybridized to each other.Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6, which describes aqueous and non-aqueousmethods, either of which can be used. Another preferred, non-limitingexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45_C, followed by one ormore washes in 2.0×SSC at 50° C. (low stringency) or 0.2×SSC, 0.1% SDSat 50-65_C (high stringency). Another preferred example of stringenthybridization conditions are hybridization in 6× sodiumchloride/sodiumcitrate (SSC) at about 45□C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50□C. Another example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45□C., followed by one or more washes in 0.2×SSC,0.1% SDS at 55□C. A further example of stringent hybridizationconditions are hybridizationin 6× sodium chloride/sodium citrate (SSC)at about 45□C., followed by one or more washes in 0.2×SSC, 0.1% SDS at60□C. Preferably, stringent hybridization conditions are hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 45□C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 65□C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65□C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65□C. In one embodiment, anisolated nucleic acid molecule of the invention that hybridizes understringent conditions to the sequence of SEQ ID NOs: 1, 3, 24, 26, 27,29, 31, 33, 35, 37, 39, 41, 43 or 45, or complement thereof, correspondsto a naturally-occurring nucleic acid molecule.

The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a yeast two hybrid assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may be,e.g., protein-protein, protein-nucleic acid, protein-small molecule, ornucleic acid-small molecule in nature.

The term “modulation” as used herein refers to both upregulation, i.e.,stimulation, and downregulation, e.g., suppression, of a response.

The term “mutated gene” refers to an allelic form of a gene, which iscapable of altering the phenotype of a subject having the mutated generelative to a subject which does not have the mutated gene. If a subjectmust be homozygous for this mutation to have an altered phenotype, themutation is said to be recessive. If one copy of the mutated gene issufficient to alter the genotype of the subject, the mutation is said tobe dominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous (for that gene) subject, the mutation is said to beco-dominant. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein).

The “non-human animals” of the invention include mammalians such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant is expressed in some but not all cells of the animal. Theterm “tissue-specific chimeric animal” indicates that one of therecombinant Delta3 genes is present and/or expressed or disrupted insome tissues but not others.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA. An “isolated” nucleic acid molecule is one which is separated fromother nucleic acid molecules which are present in the natural source ofthe nucleic acid molecule. Preferably, an “isolated” nucleic acidmolecule is free of sequences (preferably protein encoding sequences)which naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. In other embodiments, the“isolated” nucleic acid is free of intron sequences. For example, invarious embodiments, the isolated nucleic acid molecule preferablyincludes no more than 10 kilobases (kb), and more preferably, containsless than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, viral material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

The terms “protein”, “polypeptide” and “peptide” are used interchangablyherein. The term “substantially free of other cellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations ofDelta3 polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified or isolated preparations byusing a cloned gene as described herein. By “purified” or “isolated,” itis meant, when referring to a protein of the invention, that theindicated molecule is present in the substantial absence of otherbiological macromolecules, such as other proteins. The term “purified”or “isolated” as used herein preferably means at least 80% by dryweight, more preferably in the range of 95-99% by weight, and mostpreferably at least 99.8% by weight, of biological macromolecules of thesame type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than about 5000,can be present). The term “pure” or “isolated” as used herein preferablyhas the same numerical limits as “purified” or “isolated” immediatelyabove. “Isolated” and “purified” do not encompass either naturalmaterials in their native state or natural materials that have beenseparated into components (e.g., in an acrylamide gel) but not obtainedeither as pure (e.g., lacking contaminating proteins, or chromatographyreagents such as denaturing agents and polymers, e.g., acrylamide oragarose) substances or solutions. In preferred embodiments, purified orisolated Delta3 preparations will lack any contaminating proteins fromthe same animal from which Delta3 is normally produced, as can beaccomplished by recombinant expression of, for example, a human Delta3protein in a non-human cell.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such ascells of cardiac, hepatic or pancreatic origin, neuronal cells, orimmune cells. The term also covers so-called “leaky” promoters, whichregulate expression of a selected DNA primarily in one tissue, but causeexpression in other tissues as well.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. In certainembodiments, transcription of one of the recombinant Delta3 genes isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of Delta3 proteins.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a Delta3 polypeptideor, in the case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the Delta3 protein isdisrupted.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the Delta3 polypeptides, or an antisensetranscript thereto), which is partly or entirely heterologous, i.e.,foreign, to the transgenic animal or cell into which it is introduced,or, is homologous to an endogenous gene of the transgenic animal or cellinto which it is introduced, but which is designed to be inserted, or isinserted, into the animal's genome in such a way as to alter the genomeof the cell into which it is inserted (e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in a knockout). A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons, that may be necessary for optimal expression of a selectednucleic acid.

A “transgenic animal” refers to any non-human animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid (“transgene”)introduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classical cross-breeding, or invitro fertilization, but rather is directed to the introduction of arecombinant DNA molecule. This molecule may be integrated within achromosome, or it may be extrachromosomally replicating DNA. In thetypical transgenic animals described herein, the transgene causes cellsto express a recombinant form of one of the Delta3 proteins, e.g.,either agonistic or antagonistic forms. However, transgenic animals inwhich the recombinant Delta3 gene is silent are also contemplated, asfor example, the FLP or CRE recombinase dependent constructs describedbelow. Moreover, “transgenic animal” also includes those recombinantanimals in which gene disruption of one or more Delta3 genes is causedby human intervention, including both recombination and antisensetechniques. A “homologous recombinant animal” is a non-human animal,preferably a mammal, more preferably a mouse, in which an endogenousgene has been altered by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which become known in the art subsequentlyhereto.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

FTHMA-070 and T85

The present invention is based, in part, on the discovery of a cDNAmolecule encoding human FTHMA-070, a member of the TNF receptorsuperfamily.

A nucleotide sequence encoding a human FTHMA-070 protein is shown in SEQID NO:53 and SEQ ID NO:55 (open reading frame only). A predicted aminoacid sequence of FTHMA-070 protein is also shown in SEQ ID NO:54.

The FTHMA-070 cDNA of SEQ ID NO:53, which is approximately 2133nucleotides long including untranslated regions, encodes a 401 aminoacid protein.

Human FTHMA-070 is one member of a family of molecules (the “FTHMA-070family”) having certain conserved structural and functional features.The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain and havingsufficient amino acid or nucleotide sequence identity as defined herein.Such family members can be naturally occurring and can be from eitherthe same or different species. For example, a family can contain a firstprotein of human origin and a homologue of that protein of murineorigin, as well as a second, distinct protein of human origin and amurine homologue of that protein. Members of a family may also havecommon functional characteristics.

As used interchangeably herein a “FTHMA-070 activity”, “biologicalactivity of FTHMA-070” or “functional activity of FTHMA-070”, refers toan activity exerted by a FTHMA-070 protein, polypeptide or nucleic acidmolecule on a FTHMA-070 responsive cell as determined in vivo, or invitro, according to standard techniques. A FTHMA-070 activity can be adirect activity, such as an association with or an enzymatic activity ona second protein or an indirect activity.

Accordingly, another embodiment of the invention features isolatedFTHMA-070 proteins and polypeptides having a FTHMA-070 activity.

Yet another embodiment of the invention features FTHMA-070 moleculeswhich contain a signal sequence. Generally, a signal sequence (or signalpeptide) is a peptide containing about 20 amino acids which occurs atthe extreme N-terminal end of secretory and integral membrane proteinsand which contains large numbers of hydrophobic amino acid residues andserves to direct a protein containing such a sequence to a lipidbilayer.

The present invention is also based, in part, on the discovery of a cDNAmolecule encoding human T85, a protein which appears to be a secreted(non-membrane bound) form of human Robo protein, a protein which is anerve axon guidance receptor.

A nucleotide sequence encoding a human T85 protein is shown in SEQ IDNO:57 and SEQ ID NO:59 (open reading frame only). A predicted amino acidsequence of FTHMA-070 protein is also shown in SEQ ID NO:58.

The FTHMA-070 cDNA of SEQ ID NO:57, which is approximately 4291nucleotides long including untranslated regions, encodes a 753 aminoacid protein. Human T85 is one member of a family of molecules (the “T85family”) having certain conserved structural and functional features.The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain and havingsufficient amino acid or nucleotide sequence identity as defined herein.Such family members can be naturally occurring and can be from eitherthe same or different species. For example, a family can contain a firstprotein of human origin and a homologue of that protein of murineorigin, as well as a second, distinct protein of human origin and amurine homologue of that protein. Members of a family may also havecommon functional characteristics.

In one embodiment, a T85 protein includes a fibronectin type III domainhaving at least about 65%, preferably at least about 75%, and morepreferably about 85%, 95%, or 98% amino acid sequence identity to afibronectin type III domain of SEQ ID NO:61, or 62.

In one embodiment, a T85 protein includes an Ig superfamily domainhaving at least about 65%, preferably at least about 75%, and morepreferably about 85%, 95%, or 98% amino acid sequence identity to an Igsuperfamily domain of SEQ ID NO:63, 64, 65, 66, or 67.

Preferred T85 polypeptides of the present invention have an amino acidsequence sufficiently identical to a sequence identity to a fibronectintype III domain of SEQ ID NO:61 or 62 or an Ig superfamily domain of SEQID NO:63, 64, 65, 66, or 67. As used herein, the term “sufficientlyidentical” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences have acommon structural domain and/or common functional activity. For example,amino acid or nucleotide sequences which contain a common structuraldomain having about 65% identity, preferably 75% identity, morepreferably 85%, 95%, or 98% identity are defined herein as sufficientlyidentical.

As used interchangeably herein a “T85 activity”, “biological activity ofT85” or “functional activity of T85”, refers to an activity exerted by aT85 protein, polypeptide or nucleic acid molecule on a T85 responsivecell as determined in vivo, or in vitro, according to standardtechniques. A T85 activity can be a direct activity, such as anassociation with or an enzymatic activity on a second protein.

Accordingly, another embodiment of the invention features isolated T85proteins and polypeptides having a T85 activity.

Yet another embodiment of the invention features T85 molecules whichcontain a signal sequence. Generally, a signal sequence (or signalpeptide) is a peptide containing about 20 amino acids which occurs atthe extreme N-terminal end of secretory and integral membrane proteinsand which contains large numbers of hydrophobic amino acid residues andserves to direct a protein containing such a sequence to a lipidbilayer.

TANGO 77

The polypeptide cytokine interleukin-1 (IL-1) is a critical mediator ofinflammatory and overall immune response. To date, three members of theIL-1 family, IL-1α, IL-1β and IL-1ra (Interleukin-1 receptor antagonist)have been isolated and cloned. IL-1α and IL-1β are proinflammatorycytokines which elicit biological responses, whereas IL-1ra is anantagonist of IL-1α and IL-18 activity. Two distinct cell-surfacereceptors have been identified for these ligands, the type 1 IL-1receptor (IL-1RtI) and type II IL-1 receptor (IL-1RtII). Recent resultssuggest that the IL-1 RtI is the receptor responsible for transducing asignal and producing biological effects.

While inflammation is an important homeostatic mechanism, aberrantinflammation has the potential for inducing damage to the host. ElevatedIL-1 levels are known to be associated with a number of diseasesparticularly autoimmune diseases and inflammatory disorders. SinceII-1ra is a naturally occurring inhibitor of IL-1, IL-1ra can be used tolimit the aberrant and potentially deleterious effects of IL-1. Inexperimental animals, pretreatment with IL-1ra has been shown to preventdeath resulting from lipopolysaccharide-induced sepsis. The relativeabsence of IL-1ra has also been suggested to play a role in humaninflammatory bowel disease.

The present invention is based on the discovery of a cDNA moleculeencoding human Tango-77, a member of the cytokine superfamily. The cDNAmolecule encoding human Tango-77 has three possible open reading frames.The three possible nucleotide open reading frames for human Tango-77protein are shown in SEQ ID NO:73, SEQ ID NO:76 and SEQ ID NO:80. Thepredicted amino acid sequence for the three possible Tango-77 immatureproteins are shown in SEQ ID NO:72, SEQ ID NO:77 or SEQ ID NO:81 andthree possible mature proteins are shown in SEQ ID NO:75, SEQ ID NO:79and SEQ ID NO:83.

The Tango-77 cDNA of SEQ ID NO:71, which is approximately 989nucleotides long including untranslated regions, encodes a protein aminoacid having a molecular weight of approximately 19 kDa, 18 kDa, or 14.9KDa (excluding post-translational modifications) and the possible matureform of the protein has a molecular weight of 13 kDa. A plasmidcontaining a cDNA encoding human Tango-77 (with the cDNA insert name ofOf fthx077) was deposited with American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209 on Jul. 2, 1998 andassigned Accession Number 98807. This deposit will be maintained underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Thisdeposit was made merely as a convenience for those of skill in the artand is not an admission that a deposit is required under 35 U.S.C. §112.

Human Tango-77 is one member of a family of molecules (the “Tango-77family”) having certain conserved structural and functional features.The term “family,” when referring to the protein and nucleic acidmolecules of the invention, is intended to mean two or more proteins ornucleic acid molecules having a common structural domain and havingsufficient amino acid or nucleotide sequence identity as defined herein.Such family members can be naturally occurring and can be from eitherthe same or different species. For example, a family can contain a firstprotein of human origin and a homologue of that protein of murineorigin, as well as a second, distinct protein of human origin and amurine homologue of that protein. Members of a family may also havecommon functional characteristics.

As used interchangeably herein a “Tango-77 activity”, “biologicalactivity of Tango-77” or “functional activity of Tango-77”, refers to anactivity exerted by a Tango-77 protein, polypeptide or nucleic acidmolecule on a Tango-77 responsive cell as determined in vivo, or invitro, according to standard techniques. A Tango-77 activity can be adirect activity, such as an association with a second protein, or anindirect activity, such as a cellular signaling activity mediated byinteraction of the Tango-77 protein with a second protein. In apreferred embodiment, a Tango-77 activity includes at least one or moreof the following activities: (i) the ability to interact with proteinsin the Tango-77 signalling pathway (ii) the ability to interact with aTango-77 ligand or receptor; or (iii) the ability to interact with anintracellular target protein, (iv) the ability to interact with aprotein involved in inflammation, and (v) the ability to bind the IL-1receptor.

Accordingly, another embodiment of the invention features isolatedTango-77 proteins and polypeptides having a Tango-77 activity.

Yet another embodiment of the invention features Tango-77 moleculeswhich contain a signal sequence. Generally, a signal sequence (or signalpeptide) is a peptide containing about 21 to 63 amino acids which occursat the extreme N-terminal end of a secretory protein. The nativeTango-77 signal sequence (SEQ ID NO:74, SEQ ID NO:78, or SEQ ID NO:82)can be removed and replaced with a signal sequence from another protein.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of Tango-77 can be increased through use of a heterologoussignal sequence. For example, the gp67 secretory sequence of thebaculovirus envelope protein can be used as a heterologous signalsequence. Alternatively, the native Tango-77 signal sequence can itselfbe used as a heterologous signal sequence in expression systems, e.g.,to facilitate the secretion of a protein of interest.

SPOIL

Interleukin-1 (IL-1) is a multifunctional cytokine which comprises afamily of two polypeptides, IL-1α and IL-1β, with a wide spectrum ofactivities. IL-1α and IL-1β have been found to possess inflammatory,metabolic, physiologic, hematopoeitic and immunologic properties.Although both forms of IL-1 are distinct gene products, they recognizethe same cell surface receptors (i.e. IL-1 receptors, IL-1RtI andIL-1RtII).

Besides skin keratinocytes, some epithelial cells and certain cells inthe central nervous system, significant amounts of mRNA encoding IL-1are not observed in most other healthy cells. However, IL-1 productionis dramatically increased by a variety of cells in response toinfection, microbial toxins, inflammatory agents, products of activatedlymphocytes, complement and clotting components. In addition, IL-1 hasbeen recognized as a prototype of proinflammatory cytokines in that itinduces the expression of a variety of genes and the synthesis ofseveral proteins that in turn, induce acute and chronic inflammation.Thus, circulating IL-1 has been implicated in various disease statesincluding sepsis, rheumatoid arthritis, stroke and diabetes. Dinarello(1991) Blood 77(8):1627-1652.

In addition, IL-1 has been shown to regulate bone reabsorption and boneformation with its major activity in bone metabolism being osteoclastactivation. See Gowen et al. (1983) Nature 306:378-380. In fact, IL-1has been reported to be a potent stimulator of bone reabsorption and hasalso been reported to increase prostaglandin synthesis in bone. Lorenzoet al. (1987) Endocrinology 121:1164-1170.

A naturally-occurring, secreted inhibitor of IL-1 which specificallyinhibits IL-1 activity has also been identified. Carter et al. (1990)Nature 344:633. This protein, called IL-1 receptor antagonist protein(IL-1ra), has been shown to compete with the binding of IL-1 to itssurface receptors. Thus, significant interest has arisen inadministering IL-1ra to block the activity of IL-1 in various diseasesincluding septic shock (Ohlsson et al. (1990) Nature 348:550-556),immune complex-induced colitis (Cominelli (1990) J. Clin. Invest.86:972-979), acute myelogenous leukemia (Rambaldi et al. (1990) Blood76:114-120) and osteoporosis (Pacifici et al. (1993) J. Clin.Endocrinol. Metab. 77:1135-1141). Further research has indicated thatthe secreted form of IL-1ra is, in fact, a member of a family of IL-1raproteins, at least three of which are intracellular proteins (Haskill etal. (1991) Proc. Natl. Acad. Sci. USA 88:3681-3685; Muzio et al. (1995)J. Exp. Med. 182:623-628; and Weissbach et al. (1998) Biochem. Biophys.Res. Comm. 244:91-95. The family members are alternatively splicedisoforms of the IL-ra gene which consists of at least seven exons. Atruncated form of the fourth exon is produced as a result of an internalsplice acceptor site, resulting in the secreted isoform.

The present invention is based on the discovery of novel moleculeshaving homology to members of the IL-1 receptor antagonist (IL-1ra)family, referred to herein as SPOIL protein and nucleic acid molecules.The SPOIL proteins and nucleic acid molecules comprise a family ofmolecules having certain conserved structural and functional features.The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more protein ornucleic acid molecules having a common structural domain and havingsufficient amino acid or nucleotide sequence identity as defined herein.Such family members can be naturally occurring and can be from eitherthe same or different species. For example, a family can contain a firstprotein of human origin, as well as other, distinct proteins of humanorigin or alternatively, can contain homologues of non-human origin(e.g., mouse). Members of a family may also have common functionalcharacteristics.

For example, an isolated protein of the invention, preferably a SPOILprotein, is identified based on the presence of at least one “IL-1signature domain” in the protein or corresponding nucleic acid molecule.As used herein, the term “IL-1 signature domain” refers to a proteindomain which contains a conserved motif of a SPOIL protein member (orIL-1ra or IL-1 family member) and is at least about 10-30 amino acidresidues, preferably about 15-25 amino acid residues, more preferablyabout 17-24 amino acid residues, more preferably 19-23 amino acidresidues, and more preferably 21-22 amino acid residues in length. AnIL-1 signature domain includes the following amino acid motif:Xaa₁-Xaa₂-S-Xaa₃-Xaa₄-Xaa₅-P-Xaa₆-Xaa₇-Xaa₉-Xaa₉-Xaa₁₀-Xaa_(n)-Xaa₁₁,wherein Xaa₁, Xaa₂, Xaa₄, Xaa₅, Xaa₆, Xaa₇, and Xaa_(n) represents anyamino acid residue; Xaa₃ is alanine (A), serine (S), leucine (L) orvaline (V); Xaa₉ is phenylalanine (F), tyrosine (Y), leucine (L),isoleucine (I) or valine (V); Xaa₉ is either leucine (L) or isoleucine(I); Xaa₁₀ is serine (S), cysteine (C) or alanine (A); Xaa₁₁ is leucine(L), isoleucine (I), valine (V) or methionine (M); and n is about 5-25amino acid residues, more preferably about 6-18 amino acid residues, andmore preferably about 6-15 amino acid residues (SEQ ID NO:107). In oneembodiment, an IL-1 signature domain includes the following amino acidmotif: L-Xaa₁-S-V-Xaa₂-Xaa₃-P-Xaa₄-Xaa₅-Xaa_(n)-I, wherein Xaarepresents any amino acid, and n is about 5-25 amino acid residues, morepreferably about 6-18 amino acid residues, and more preferably about6-15 amino acid residues (SEQ ID NO:108). Preferably, the IL-1 signaturedomain includes the following amino acid sequence:L-Xaa₁-S-V-Xaa₂-Xaa₃-P-Xaa₄-Xaa₅-Xaa_(n)-I, wherein Xaa₁ is eitherthreonine (T) or glutamic acid (E); Xaa₂ is either alanine (A) orglutamic acid (E); and Xaa₅ is either tryptophan (W) or leucine (L) (SEQID NO:111). In another embodiment, an IL-1 signature domain includes thefollowing amino acid sequence motif:F-Xaa₁-S-A-Xaa₂-Xaa₃-P-Xaa₄-Xaa₅-Xaa_(n)-L, wherein Xaa represents anyamino acid, and n is about 5-25 amino acid residues, more preferablyabout 6-18 amino acid residues, and more preferably about 6-15 aminoacid residues (SEQ ID NO:94). Preferably, the IL-1 signature domainincludes the following amino acid sequence:F-Xaa₁-S-A-Xaa₂-Xaa₃-P-Xaa₄-Xaa₅-Xaa_(n)-L, wherein Xaa₁ is eitherthreonine (T) or glutamic acid (E); Xaa₂ is either alanine (A) orglutamic acid (E); and Xaa₅ is either tryptophan (W) or leucine (L) (SEQID NO:95). In yet another embodiment, the IL-1 signature domain is atleast about 10-30 amino acid residues in length, preferably 15-25 aminoacid residues in length, preferably 17-24 amino acid residues, 19-23amino acid residues or more preferably 21-22 amino acid residues inlength and has at least about 30-60% identity, preferably at least about35-55% identity, more preferably at least about 40-50% identity, andmore preferably at least about 46-49% identity with an IL-1 signaturedomain of a protein of the invention having an amino acid sequence asset forth in SEQ ID NO:90 (e.g., amino acid residues 58-80), SEQ IDNO:102 (e.g., amino acid residues 130-151), SEQ ID NO:105 (e.g., aminoacid residues 169-190), or SEQ ID NO:113 (e.g., amino acid residues120-142).

In a preferred embodiment, a protein of the invention, preferably aSPOIL protein, contains an IL-1 signature domain of SEQ ID NO:90 (e.g.,amino acid residues 58-80), SEQ ID NO:102 (e.g., amino acid residues130-151), SEQ ID NO:105 (e.g., amino acid residues 169-190), or SEQ IDNO:113 (e.g., amino acid residues 120-142).

In another embodiment of the invention, a SPOIL protein is identifiedbased on the presence of at least one “SPOIL signature motif” in theprotein or corresponding nucleic acid molecule. As used herein, the term“SPOIL signature motif” includes an amino acid sequence which containsamino acid residues that are conserved among SPOIL family members. Inone embodiment, a SPOIL signature motif, referred to herein as a “shortSPOIL signature motif”, includes an amino acid sequence at least about35-55 amino acid residues, preferably about 38-50 amino acid residues,more preferably about 40-48 amino acid residues, more preferably 42-46amino acid residues, and more preferably 44 amino acid residues inlength and having the following amino acid sequence:Q-Xaa₁-Xaa₂-E-Xaa₃-Xaa₄-1-M-Xaa₅-Xaa₆-Y-Xaa₇-Xaa₈-Xaa₉-E-P-V-K-Xaa₁₀-Xaa₁₁-L-F-Y-Xaa₁₂-Xaa₁₃-K-Xaa₁₄-G-Xaa₁₅-T-S-T-Xaa₁₆-E-S-Xaa₁₇-A-F-P-Xaa₁₈-W-F-1-A,wherein Xaa₁₋₁₈ is any amino acid (set forth in SEQ ID NO:109).Accordingly, preferred proteins include the conserved amino acidresidues of the above-recited SPOIL signature motif. Proteins includingat least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43conserved amino acid residues of the above-recited SPOIL signature motifare also considered to be within the scope of the present invention.

In another embodiment, a SPOIL signature motif, referred to herein as a“long SPOIL signature motif” includes an amino acid sequence of at leastabout 58-78 amino acid residues, preferably about 61-74 amino acidresidues, more preferably about 63-72 amino acid residues, morepreferably 65-70 amino acid residues, and more preferably 67-68 aminoacid residues in length and having the following amino acid sequence:Q-Xaa₁-Xaa₂-E-Xaa₃-Xaa₄-1-M-Xaa₅-Xaa₆-Y-Xaa₇-Xaa₈-Xaa₉-E-P-V-K-Xaa₁₀-Xaa₁₁-L-F-Y-Xaa₁₂-Xaa₁₃-K-Xaa₁₄-G-Xaa₁₅-T-S-T-Xaa₁₆-E-S-Xaa₁₇-A-F-P-Xaa₁₈-W-F-1-A-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-P-Xaa₂₆-1-L-T-Xaa₂₇-E-L-G-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-T-Xaa₃₂-F-E,wherein Xaa_(1-24 and 26-32) is any amino acid Xaa₂₅ is no amino acid orany amino acid (set forth in SEQ ID NO:110). A preferred proteinincludes the conserved amino acid residues of the above-recited SPOILsignature motif. Proteins including at least 56, 57, 58, 59, 60, 61, 62,63, 64, 65, or 66 conserved amino acid residues of the above-recitedSPOIL signature motif are also considered to be within the scope of thepresent invention.

Table III depicts the conserved amino acid residues of the SPOILsignature motifs set forth in SEQ ID NO:109 and SEQ ID NO:110. Theconserved amino acid residues are numbered according to their positionin the SPOIL signature motif as well as by their relative amino acidposition in each of murine SPOIL-I, murine SPOIL-II, human SPOIL-I andhuman SPOIL-II. As used herein, the amino acid residues in each of theSPOIL proteins “correspond to” the relative amino acid residues in aSPOIL signature motif.

TABLE III corre- corre- corre- corre- Residue in sponding spondingsponding sponding SPOIL residue in residue in residue in residue insignature motif muSPOIL-I muSPOIL-II huSPOIL-I huSPOIL-II Gln1 Gln26Gln88 Gln98 Gln137 Glu4 Glu29 Glu91 Glu101 Glu140 Ile7 Ile32 Ile94Ile104 Ile143 Met8 Met33 Met95 Met105 Met144 Tyr11 Tyr36 Tyr98 Tyr108Tyr147 Glu15 Glu40 Glu102 Glu112 Glu151 Pro16 Pro41 Pro103 Pro113 Pro152Val17 Val42 Val104 Val114 Val153 Lys18 Lys43 Lys105 Lys115 Lys154 Leu21Leu46 Leu108 Leu118 Leu157 Phe22 Phe47 Phe109 Phe119 Phe158 Tyr23 Tyr48Tyr110 Tyr120 Tyr159 Lys26 Lys51 Lys113 Lys123 Lys162 Gly28 Gly53 Gly115Gly125 Gly164 Thr30 Thr55 Thr117 Thr127 Thr166 Ser31 Ser56 Ser118 Ser128Ser167 Thr32 Thr57 Thr119 Thr129 Thr168 Glu34 Glu59 Glu121 Glu131 Glu170Ser35 Ser60 Ser122 Ser132 Ser171 Ala37 Ala62 Ala124 Ala134 Ala173 Phe38Phe63 Phe125 Phe135 Phe174 Pro39 Pro64 Pro126 Pro136 Pro175 Trp41 Trp66Trp128 Trp138 Trp177 Phe42 Phe67 Phe129 Phe139 Phe178 Ile43 Ile68 Ile130Ile140 Ile179 Ala44 Ala69 Ala131 Ala141 Ala180 Pro51 Pro77 Pro139 Pro148Pro187 Ile53 Ile79 Ile141 Ile150 Ile189 Leu54 Leu80 Leu142 Leu151 Leu190Thr55 Thr81 Thr143 Thr152 Thr191 Glu57 Glu83 Glu145 Glu154 Glu193 Leu58Leu84 Leu146 Leu155 Leu194 Gly59 Gly85 Gly147 Gly156 Gly195 Thr64 Thr90Thr152 Thr161 Thr200 Phe66 Phe92 Phe154 Phe163 Phe202 Glu67 Glu93 Glu155Glu164 Glu203

Another embodiment of the invention features proteins having a “SPOILunique domain”. As used herein, a “SPOIL unique domain” is at leastabout 134-150 amino acid residues in length and has at least about45-50% identity with amino acid residues 66-206 of SEQ ID NO:105. Inanother embodiment, the SPOIL unique domain is at least about 136-148amino acid residues, preferably about 138-146 amino acid residues, morepreferably 140-144 amino acid residues, and more preferably 141, 142, or143 amino acid residues in length and has at least about 55-60%,preferably about 65-70%, and more preferably about 75%, 80%, 85%, 90%,95%, 98%, 99%, or 100% identity with amino acid residues 66-206 of SEQID NO:105. In a preferred embodiment, the SPOIL unique domain is fromabout amino acid residues 66-206 of human SPOIL-II shown in SEQ IDNO:105. In another preferred embodiment, the SPOIL unique domain is fromabout amino acid residues 27-167 of human SPOIL-I shown in SEQ IDNO:102. In yet another preferred embodiment, the SPOIL unique domain isfrom about amino acid residues 17-158 of murine SPOIL-II shown in SEQ IDNO:113.

Another embodiment of the invention features proteins having a “SPOILC-terminal unique domain”. As used herein, a “SPOIL C-terminal uniquedomain” is at least about 58-78 amino acid residues in length and has atleast about 45-50% identity with amino acid residues 137-203 of SEQ IDNO:105. In another embodiment, the SPOIL C-terminal unique domain is atleast about 61-74 amino acid residues, preferably about 63-72 amino acidresidues, more preferably 65-70 amino acid residues, and more preferably67-68 amino acid residues in length and has about 55-60%, preferablyabout 65-70%, and more preferably about 75%, 80%, 85%, 90%, 95%, 98%,99%, or 100% identity with amino acid residues 137-203 of SEQ ID NO:105.In one embodiment, the C-terminal unique domain is located within theC-terminal 70 amino acids of the full-length protein, preferably withinthe C-terminal 80 amino acid residues of the protein, more preferablywithin the C-terminal 90 amino acid residues of the protein, and evenmore preferably within the C-terminal 100, 120, 140, 160 or 180 aminoacid residues of the full-length protein. In a preferred embodiment, theSPOIL C-terminal unique domain is from about amino acid residues 137-203of human SPOIL-II shown in SEQ ID NO:105. In another preferredembodiment, the SPOIL C-terminal unique domain is from about amino acidresidues 98-164 of human SPOIL-I shown in SEQ ID NO:102. In anotherpreferred embodiment, the SPOIL C-terminal unique domain is from aboutamino acid residues 26-93 of murine SPOIL-I shown in SEQ ID NO:90. Inyet another preferred embodiment, the SPOIL C-terminal unique domain isfrom about amino acid residues 88-155 of murine SPOIL-II shown in SEQ IDNO:113.

Another embodiment of the invention features a protein of the invention,preferably a SPOIL protein, which contain a signal sequence. As usedherein, a “signal sequence” refers to a peptide containing about 17amino acids which occurs at the N-terminus of secretory proteins andwhich contains a large number of hydrophobic amino acid residues. Forexample, a signal sequence contains at least about 13-22 amino acidresidues, preferably about 15-20 amino acid residues, more preferablyabout 16-19 amino acid residues, and more preferably about 17 amino acidresidues, and has at least about 35-65%, preferably about 38-50%, andmore preferably about 40-45% hydrophobic amino acid residues (e.g.,Valine, Leucine, Isoleucine or Phenylalanine). Such a “signal sequence”,also referred to in the art as a “signal peptide”, serves to direct aprotein containing such a sequence to a lipid bilayer. For example, inone embodiment, a SPOIL protein contains a signal sequence containingabout amino acids 1-17 of SEQ ID NO:90.

In yet another embodiment, a protein of the invention, preferably aSPOIL protein, encodes a mature protein. As used herein, the term“mature protein” refers to a protein of the invention, preferably aSPOIL protein, from which the signal peptide has been cleaved. In anexemplary embodiment, a mature SPOIL protein contains amino acidresidues 1 to 81 of SEQ ID NO:93. In a preferred embodiment, a SPOILprotein is a mature SPOIL protein which includes an IL-1 signaturedomain. In yet another embodiment, a SPOIL protein is a mature proteinwhich includes a SPOIL signature motif and/or a SPOIL C-terminal uniquedomain.

Preferred proteins of the present invention, preferably SPOIL proteins,have an amino acid sequence sufficiently homologous to the amino acidsequence of SEQ ID NO:90; SEQ ID NO:93; SEQ ID NO:102, SEQ ID NO:105,SEQ ID NO:113, the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984. As used herein, the term “sufficientlyhomologous” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences sharecommon structural domains or motifs and/or a common functional activity.For example, amino acid or nucleotide sequences which share commonstructural domains have at least 45% or 50% identity, preferably 60%identity, more preferably 70%-80%, and even more preferably 90-95%identity across the amino acid sequences of the domains and contain atleast one and preferably two structural domains or motifs, are definedherein as sufficiently homologous. Furthermore, amino acid or nucleotidesequences which share at least 45% or 50%, preferably 60%, morepreferably 70-80%, or 90-95% identity and share a common functionalactivity are defined herein as sufficiently homologous.

As used interchangeably herein a “SPOIL activity”, “biological activityof SPOIL” or “functional activity of SPOIL”, refers to an activityexerted by a SPOIL protein, polypeptide or nucleic acid molecule on aSPOIL responsive cell as determined in vivo, or in vitro, according tostandard techniques. In one embodiment, a SPOIL activity is a directactivity, such as an association with a target protein, preferably aSPOIL target molecule (e.g., a cell-surface or internalized IL-1 orSPOIL receptor). In another embodiment, a SPOIL activity is an indirectactivity, such as inhibiting the synthesis or activity of a secondprotein (e.g., a protein of a signal pathway). In a preferredembodiment, a SPOIL activity is at least one or more of the followingactivities: (i) interaction of a SPOIL protein in the extracellularmilieu with a protein molecule on the surface of the same cell whichsecreted the SPOIL protein molecule (e.g., a SPOIL receptor or IL-1receptor); (ii) interaction of a SPOIL protein in the extracellularmilieu with a protein molecule on the surface of a different cell fromthat which secreted the SPOIL protein molecule (e.g., a SPOIL receptoror IL-1 receptor); (iii) complex formation between a SPOIL protein and acell-surface receptor; (iv) interaction of a SPOIL protein with a targetmolecule in the extracellular milieu (e.g., a soluble target molecule);(v) interaction of the SPOIL protein with an intracellular targetmolecule (e.g., interaction with an internalized or endocytosed receptoror ligand-coupled receptor); and (vi) complex formation with at leastone, preferably two or more, intracellular target molecules.

In yet another preferred embodiment, a SPOIL activity is at least one ormore of the following activities: (1) modulating, for example,antagonizing a signal transduction pathway (e.g., an IL-1-dependent orSPOIL-dependent pathway; (2) modulating cytokine production and/orsecretion (e.g., production and/or secretion of a proinflammatorycytokine); (3) modulating lymphokine production and/or secretion; (4)modulating production of adhesion molecules and/or cellular adhesion;(5) modulating expression or activity of nuclear transcription factors;(6) modulating secretion of IL-1; (7) competing with IL-1 to bind anIL-1 receptor; (8) competing with a SPOIL protein (e.g., a SPOIL-I orSPOIL-II family member) to bind a SPOIL receptor; (9) modulating nucleartranslocation of internalized IL-1 or SPOIL receptor or ligand-complexedreceptor; (10) modulating cell proliferation, development ordifferentiation, for example, IL-1-stimulated or a SPOILprotein-stimulated proliferation, development or differentiation (e.g.,of an epithelial cell, for example, a squamous epithelial cell of theesophagus, or of a skin cell, e.g., a keratinocyte); (11) modulatingcell proliferation, development or differentiation of an osteogenic cell(e.g., of an osteoclast precursor cell, osteoclast and/or osteoblast);(12) modulating function of an osteogenic cell (e.g., osteoblast and/orosteoclast function); (13) modulating bone formation, bone metabolismand/or bone homeostasis (e.g., inhibiting bone resorption); (14)modulating cellular immune responses; (15) modulating cytokine-mediatedproinflammatory actions (e.g., inhibiting acute phase protein synthesisby hepatocytes, fever, and/or prostaglandin synthesis, for example PGE₂synthesis); and (16) promoting and/or potentiating wound healing.

The present invention is based, at least in part, on the discovery of afamily of SPOIL proteins (e.g., SPOIL-I and SPOIL-II proteins) sharingcertain conserved structural features (e.g., a SPOIL signature motif, anIL-1 signature domain, a SPOIL C-terminal unique domain). Moreover, ithas been discovered that SPOIL proteins exist as multiple isoforms,presumably due to alternative splicing of one or more common genes. Forexample, SPOIL proteins having internal inserted amino acid segmentshave been identified (e.g., human SPOIL-II includes a segment of atleast 40 amino acid residues not appearing in human SPOIL-I). SPOILproteins have also been identified which may function as both secretedand intracellular molecules (e.g., murine SPOIL-I has a signal sequencewhich is not found in murine SPOIL-II). Therefore, additional SPOILfamily members can be identified based on the nucleotide and amino acidsequence information provided herein which, e.g., via alternativesplicing of genomic SPOIL sequences, have unique combinations of thestructural features defined herein. For example, secreted isoforms ofhuman SPOIL can be identified which include all, or a portion of theamino acid sequences set forth as SEQ ID NOs:102 and 105.

Moreover, SPOIL family members can be identified based on uniquenucleotide and/or amino acid sequences found in one SPOIL family memberas compared to a second SPOIL family member. For example, a comparisonbetween the nucleotide sequences of murine SPOIL-I (SEQ ID NO:89) andmurine SPOIL-II (SEQ ID NO:112) reveals that murine SPOIL-II includes afragment from nucleotides 225 to 364 that is absent from murine SPOIL-I(SEQ ID NO:89). Moreover, a comparison of the amino acid sequences ofmurine SPOIL-I (SEQ ID NO:90) and murine SPOIL-II (SEQ ID NO:113)reveals that murine SPOIL-II includes a fragment from amino acids 1 to90 that is absent from murine SPOIL-I. Accordingly, one embodiment ofthe present invention includes an isolated nucleic acid moleculeincluding nucleotides 225 to 364 of SEQ ID NO:112. In anotherembodiment, an isolated nucleic acid molecule of the present inventionincludes at least 30 contiguous nucleotides of SEQ ID NO:112 fromnucleotides 225 to 364. In another embodiment, an isolated nucleic acidmolecule of the present invention includes at least 20-140, 30-130,40-120, 50-110, 60-100, 70, 80, or 90 contiguous nucleotides of SEQ IDNO:112 from nucleotides 225 to 364. In yet another embodiment, anisolated nucleic acid molecule of the present invention has at leastabout 50% identity to nucleotides 225 to 364 of SEQ ID NO:112. In yetanother embodiment, an isolated nucleic acid molecule has at least 50%identity to at least 30 contiguous nucleotides of SEQ ID NO:112 fromnucleotides 224 to 364. In yet another embodiment, an isolated nucleicacid molecule of the invention hybridizes under stringent conditions tonucleotides 225 to 364 of SEQ ID NO:112. In yet another embodiment, anisolated nucleic acid molecule hybridizes under stringent conditions toat least 30 contiguous nucleotides of SEQ ID NO:112 from nucleotides 225to 364.

Another embodiment of the present invention pertains to a polypeptideincluding amino acids 1 to 90 of SEQ ID NO:113. In yet anotherembodiment, the polypeptide includes at least 30 contiguous amino acidsof SEQ ID NO:113 from amino acids 1 to 90 of SEQ ID NO:113. In yetanother embodiment, the polypeptide includes at least 10-90, 20-80,30-70, 40, 50 or 60 contiguous amino acids of SEQ ID NO:113 from aminoacids 1 to 90. Yet another embodiment of the invention pertains to apolypeptide having at least 50% identity to amino acids 1 to 90 of SEQID NO:113. In yet another embodiment, the polypeptide has at least 50%identity to at least 10-90, 20-80, 30-70, 40, 50 or 60 contiguous aminoacids of SEQ ID NO:113 from amino acids 1 to 90. Yet another embodimentof the present invention features isolated nucleic acid moleculesencoding any of the polypeptides described herein.

Likewise, a comparison between the nucleic acid sequences of humanSPOIL-I (SEQ ID NO:101) and human SPOIL-II (SEQ ID NO:104) reveals thathuman SPOIL-II includes a fragment from nucleotides 153 to 269 that isabsent from human SPOIL-I (SEQ ID NO:101). Moreover, a comparison of theamino acid sequences of human SPOIL-I (SEQ ID NO:102) and human SPOIL-II(SEQ ID NO:105) reveals that human SPOIL-II includes a fragment fromamino acids 19 to 58 that is absent from human SPOIL-I. Accordingly, oneembodiment of the present invention includes an isolated nucleic acidmolecule including nucleotides 153 to 269 of SEQ ID NO:112. In anotherembodiment, an isolated nucleic acid molecule of the present inventionincludes at least 30 contiguous nucleotides of SEQ ID NO:112 fromnucleotides 153 to 269. In another embodiment, an isolated nucleic acidmolecule of the present invention includes at least 20-140, 30-130,40-120, 50-110, 60-100, 70, 80, or 90 contiguous nucleotides of SEQ IDNO:104 from nucleotides 153 to 269. In yet another embodiment, anisolated nucleic acid molecule of the present invention has at leastabout 50% identity to nucleotides 153 to 269 of SEQ ID NO:104. In yetanother embodiment, an isolated nucleic acid molecule has at least 50%identity to at least 30 contiguous nucleotides of SEQ ID NO:104 fromnucleotides 224 to 364. In yet another embodiment, an isolated nucleicacid molecule of the invention hybridizes under stringent conditions tonucleotides 153 to 269 of SEQ ID NO:104. In yet another embodiment, anisolated nucleic acid molecule hybridizes under stringent conditions toat least 30 contiguous nucleotides of SEQ ID NO:104 from nucleotides 153to 269.

Another embodiment of the present invention includes a polypeptideincluding amino acids 19 to 58 of SEQ ID NO:105. In yet anotherembodiment, the invention features a polypeptide which includes at least30 contiguous amino acids of SEQ ID NO:105 from amino acids 19 to 58 ofSEQ ID NO:105. In yet another embodiment, the polypeptide includes atleast 10-90, 20-80, 30-70, 40, 50 or 60 contiguous amino acids of SEQ IDNO:105 from amino acids 19 to 58. In yet another embodiment, thepolypeptide has at least 50% identity to amino acids 19 to 58 of SEQ IDNO:105. In yet another embodiment, the polypeptide has at least 50%identity to at least 10-90, 20-80, 30-70, 40, 50 or 60 contiguous aminoacids of SEQ ID NO:105 from amino acids 19 to 58. Yet another embodimentof the present invention features isolated nucleic acid moleculesencoding any of the polypeptides described herein.

Given the existence of both secreted and intracellular SPOIL molecules(e.g., SPOIL-I and II isoforms) described herein, it will be appreciatedthat the SPOIL molecules of the present invention and modulators ofSPOIL proteins are useful, for example, in regulating cellular responsestriggered by extracellular events (e.g., by interaction of, for example,a cytokine (e.g., IL-1) or a SPOIL protein with a cell surface receptor.For example, it is known that unbalanced production of IL-1 isassociated with the pathogenesis of various inflammatory diseases.Accordingly, SPOIL proteins and/or SPOIL modulators may be useful astherapeutic agents in achieving homeostasis and ameliorating suchimbalances.

Likewise, it will be appreciated that the SPOIL molecules of the presentinvention and modulators of SPOIL proteins are useful in regulatingcytokine (e.g., IL-1) and/or SPOIL protein dependent intracellularresponses (e.g., acting as intracellular antagonists or agonists). Forexample, it is known that cytokines (e.g., IL-1) are not secreted fromcertain cell types, for example, skin cells, e.g., keratinocytes, andaccordingly, there exist a discreet subset of intracellularcytokine-dependent responses and a corresponding set of intracellularSPOIL protein-dependent activities.

Moreover, SPOIL molecules of the present invention have been found to beconstitutively expressed, for example, in epithelial cells, inparticular in the squamous epithelium of the esophagus and theepithelial lining of the mouth (e.g., murine SPOIL-II was isolated froman esophageal cDNA library). In addition, expression of SPOIL moleculescan also be induced in certain cell types and tissues. For example, thehuman SPOILs were isolated from a stimulated keratinocyte library andhuman SPOIL-I was expressed in keratinocytes induced with PMA,ionomycin, TNF and cyclohexamide. In addition, human SPOIL-I wasobserved in monocytic cells stimulated with LPS and expression ofSPOIL-I was induced in the kidneys of lippopolysaccharide (LPS)-injectedmice. Furthermore, expression has been correlated with certainproliferative disorders. For example, human SPOIL-I was found to beexpressed on human esophageal tumor samples and overexpressed insquamous cell carcinoma of the esophagus. It has further beendemonstrated that a secreted form of SPOIL (e.g., murine SPOIL-I), whenexpressed in vivo, caused impairment of osteoclast differentiationand/or function as well as evidence of impaired bone resorption (seeEXAMPLE 25).

Accordingly, in another embodiment of the invention, a SPOIL molecule orpreferably, a SPOIL modulator, is useful for regulating, preventingand/or treating at least one or more of the following diseases ordisorders: (1) inflammatory diseases and disorders including, but notlimited to, inflammation, septic shock, arthritis, intercolitis, andpneumonitis; (2) epithelial cell and/or skin diseases and disordersincluding, but not limited to proliferative disorders (e.g., skincancers including, but not limited to, melanoma, and Kaposi's sarcoma,and other epithelial cancers including squamous cell carcinoma,esophageal cancer and cancer of the mouth and/or throat); (3)bone-related and/or bone resorption disorders including, but not limitedto osteoporosis, Paget's disease, osteoarthritis, degenerativearthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia,bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) andhypercalcemia; and (4) diseases and disorders that involve the bowel andare characterized by the production of inflammation and at times,ulceration in the small or large bowel, including but not limited to,inflammatory bowl disease (IBD) (e.g., Crohn's disease, irritable bowelsyndrome (IBS) and ulcerative colitis). Moreover, it will be appreciatedthat the SPOIL molecules and SPOIL modulators are useful for thefollowing purposes: (1) regulation of bone mass (e.g., increase bonemass and/or inhibit bone loss); (2) management of bone fragility (e.g.,decrease bone fragility); and (3) prevention and/or treatment of bonepain, bone deformities, and/or bone fractures.

Another embodiment of the invention features isolated SPOIL proteins andpolypeptides having a conserved SPOIL structural feature and a SPOILactivity, as defined herein. Preferred SPOIL proteins have an IL-1signature domain and a SPOIL activity. In one embodiment, the SPOILprotein has a signal peptide, an IL-1 signature domain, and a SPOILactivity. In another preferred embodiment, the SPOIL protein has asignal peptide, an IL-1 signature domain, a SPOIL activity, and an aminoacid sequence sufficiently homologous to an amino acid sequence of SEQID NO:90; SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984.

Another embodiment of the invention features isolated SPOIL proteins andpolypeptides having a SPOIL activity, a SPOIL signature motif (short orlong form) and/or SPOIL unique domain. In another embodiment, the SPOILprotein has a SPOIL activity, a SPOIL signature motif (short or longform) and/or SPOIL C-terminal unique domain. In another preferredembodiment, the SPOIL protein has a SPOIL activity, a SPOIL signaturemotif (short or long form) and/or SPOIL C-terminal unique domain, and anamino acid sequence sufficiently homologous to an amino acid sequence ofSEQ ID NO:90; SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, the aminoacid sequence encoded by the DNA insert of the plasmid deposited withATCC as Accession Number 98883, or the amino acid sequence encoded bythe DNA insert of the plasmid deposited with ATCC as Accession Number98984. The above-described SPOIL proteins can further include an IL-1signature domain as described herein.

In a particularly preferred embodiment, the SPOIL protein and nucleicacid molecules of the present invention are human SPOIL molecules. Anucleotide sequence of the isolated human SPOIL-I cDNA and the predictedamino acid sequence of the human SPOIL-I protein are shown in SEQ IDNOs:101 and 102, respectively. In addition, the nucleotide sequencecorresponding to the coding region of the human SPOIL-I cDNA(nucleotides 124 to 630) is represented as SEQ ID NO:103.

The human SPOIL-I cDNA, which is approximately 1291 nucleotides inlength, encodes a protein which is approximately 169 amino acid residuesin length. A plasmid containing the full length nucleotide sequenceencoding human SPOIL-I (clone designation Epjthkf 035f11) was depositedunder the provisions of the Budapest Treaty with the American TypeCulture Collection (ATCC), presently in Manassas, Va., on Sep. 11, 1998and assigned Accession Number 98883. The human SPOIL-I protein containsan IL-1 signature domain, which can be found, for example, from aboutamino acids 130 to 151 of SEQ ID NO:102 (Leu130 to Leu151 of the humanSPOIL-I amino acid sequence). The human SPOIL-I protein further containsa SPOIL signature motif, which can be found, for example, from aboutamino acids 98-141 (short) of from about 98-164 (long) of SEQ ID NO:102(Gln98 to Ala141 or Gln98 to Glu164 of the human SPOIL-I amino acidsequence). A SPOIL C-terminal unique domain can be found in the humanSPOIL-I protein, for example, from about amino acid residues 98-164 ofSEQ ID NO:102 (Gln98 to Glu164 of the human SPOIL-I amino acidsequence).

A nucleotide sequence of the isolated human SPOIL-II cDNA and thepredicted amino acid sequence of the human SPOIL-II protein are shown inSEQ ID NOs:106 and 107, respectively. In addition, the nucleotidesequence corresponding to the coding region of the human SPOIL-II cDNA(nucleotides 98-721) is represented as SEQ ID NO:106.

The human SPOIL-II cDNA, which is approximately 1377 nucleotides inlength, encodes a protein which is approximately 208 amino acid residuesin length. A plasmid containing the full length nucleotide sequenceencoding human SPOIL-II (clone designation Epjthkf 074e01) was depositedunder the provisions of the Budapest Treaty with the American TypeCulture Collection (ATCC), presently in Manassas, Va., on Nov. 11, 1998and assigned Accession Number 98984. The human SPOIL-II protein containsan IL-1 signature domain, which can be found, for example, from aboutamino acids 169-190 of SEQ ID NO:102 (Leu169 to Leu190 of the humanSPOIL-II amino acid sequence). The human SPOIL-II protein furthercontains a SPOIL signature motif, which can be found, for example, fromabout amino acids 137-180 (short) of from about 137-203 (long) of SEQ IDNO:105 (Gln137 to Ala180 or Gln137 to Glu203 of the human SPOIL-II aminoacid sequence). A SPOIL C-terminal unique domain can be found in thehuman SPOIL-II protein, for example, from about amino acid residues137-203 of SEQ ID NO:105 (Gln137 to Glu203 of the human SPOIL-II aminoacid sequence), having 100% identity to the SPOIL C-terminal uniquedomain of human SPOIL-I.

In another embodiment, the SPOIL protein and nucleic acid molecules ofthe present invention are murine SPOIL molecules. A nucleotide sequenceof the isolated murine SPOIL-I cDNA and the predicted amino acidsequence of the murine SPOIL-I protein are shown in SEQ ID NOs:89 and90, respectively. In addition, the nucleotide sequences corresponding tothe coding region of the murine SPOIL-I cDNA (nucleotides 135-428) andthe SPOIL-I cDNA encoding the mature SPOIL-I protein are represented asSEQ ID NO:91 and SEQ ID NO:92, respectively.

The murine SPOIL-I cDNA (set forth in SEQ ID NO:89), which isapproximately 746 nucleotides in length, encodes a protein having amolecular weight of approximately 10.96 kD (with signal sequence) and9.1 kD (without signal sequence) and which is approximately 98 aminoacid residues in length (SEQ ID NO:90). The murine SPOIL-I proteincontains an IL-1 signature domain as defined herein, which can be found,for example, from about amino acids 58 to 80 of SEQ ID NO:90 and, forexample, from about amino acids 41-63 of SEQ ID NO:93. The murineSPOIL-I protein further contains a SPOIL signature motif, which can befound, for example, from about amino acids 26-69 (short) of from about26-93 (long) of SEQ ID NO:90 (Gln26 to Ala69 or Gln26 to Glu93 of themurine SPOIL-I amino acid sequence). A SPOIL C-terminal unique domaincan be found in the murine SPOIL-I protein, for example, from aboutamino acid residues 26-93 of SEQ ID NO:105 (Gln26 to Glu93 of the murineSPOIL-I amino acid sequence), having 52.2% identity to the SPOILC-terminal unique domain of human SPOIL-I. (Comparison can be madeusing, for example, the Lipman-Pearson Algorithm (Lipman and Pearson(1985) Science 227:1435-1441, with a K-tuple of 2, a Gap Penalty of 4,and a Gap Weight Penalty of 12. In addition, the murine SPOIL-I proteincan contain a signal sequence. A signal sequence can be found at least,for example, from about amino acids 1-17 of SEQ ID NO:90. The predictionof such a signal peptide can be made, for example, utilizing thecomputer algorithm SignalP (Henrik, et al. (1997) Protein Engineering10:1-6).

The entire amino acid sequence of SEQ ID NO:90 was subcloned intoretroviral vector MSCVneo (Hawley, et al. (1994) Gene Therapy 1:136-138)and used for retroviral delivery. Bone marrow infected with theretroviral vector expressing murine SPOIL-I was transplanted intoirradiated mice recipients. Bones removed from these mouse recipients,histologically, appeared thicker than the bones of control mice. Inaddition, spleen cells (i.e., a source of osteoclast progenitors) whichwere removed from mice recipients and were cultured on a bone marrowcell line, demonstrated reduced osteoclast production than the spleencells of control mice. These experiments are discussed in further detailherein.

According to in situ analysis of mouse tissues, in the tissues tested,SPOIL-I mRNA transcript is expressed almost exclusively in the squamouscell epithelium of the esophagus and in the epithelial lining of themouth. Northern blot analysis of human tissues confirms this pattern ofSPOIL expression with transcripts being detected in esophagus withexpression also likely in the trachea, among the tissues tested. Inaddition, SPOIL is also present on human esophageal tumor samples andoverexpressed in moderately differentiated squamous cell carcinoma ofthe esophagus.

A multiple sequence alignment of the amino acid sequences of murineSPOIL-I with murine IL-1ra (Swiss-Prot™ Accession No. P25085) (SEQ IDNO:98), as well as murine IL-1α (Swiss-Prot™ Accession No. P01582) (SEQID NO:99) and murine IL-1β (Swiss-Prot™ Accession No. P10749) (SEQ IDNO:100) was generated using MegAlign™ sequence alignment software (referto FIG. 4).

A nucleotide sequence of the isolated murine SPOIL-II cDNA and thepredicted amino acid sequence of the murine SPOIL-II protein are shownin SEQ ID NOs:112 and 113, respectively. In addition, the nucleotidesequences corresponding to the coding region of the murine SPOIL-II cDNA(nucleotides 96-575) is represented as SEQ ID NO:114.

The murine SPOIL-II cDNA (set forth in SEQ ID NO:112), which isapproximately 838 nucleotides in length, encodes a protein which isapproximately 160 amino acid residues in length (SEQ ID NO:113). Themurine SPOIL-II protein contains an IL-1 signature domain as definedherein, which can be found, for example, from about amino acids 120 to142 of SEQ ID NO:113. The murine SPOIL-II protein further contains aSPOIL signature motif, which can be found, for example, from about aminoacids 88-131 (short) of from about 88-155 (long) of SEQ ID NO:113 (Gln88to Ala131 or Gln88 to Glu155 of the murine SPOIL-II amino acidsequence). A SPOIL C-terminal unique domain can be found in the murineSPOIL-II protein, for example, from about amino acid residues 88-155 ofSEQ ID NO:113 (Gln88 to Glu155 of the murine SPOIL-II amino acidsequence), having 52.2% identity to the SPOIL C-terminal unique domainof human SPOIL-I.

A multiple sequence alignment of the amino acid sequences of humanSPOIL-I (corresponding to amino acid residues 1-169 of SEQ ID NO:102),human SPOIL-II (corresponding to amino acid residues 1-208 of SEQ IDNO:105), murine SPOIL-I (corresponding to amino acid residues 1-98 ofSEQ ID NO:90), and murine SPOIL-II (corresponding to amino acid residues1-160 of SEQ ID NO:113) is shown in FIG. 6.

A multiple sequence alignment of the amino acid sequences of murineSPOIL-I, murine SPOIL-II, human SPOIL-I, and human SPOIL-II with murineIL-1ra (Swiss-Prot™ Accession No. P25085) (SEQ ID NO:98), as well asmurine (Swiss-Prot™ Accession No. P01582) (SEQ ID NO:99) and murineIL-1β (Swiss-Prot™ Accession No. P10749) (SEQ ID NO:100) is shown inFIG. 7. (The alignments of FIGS. 4, 6, and 7 were generated usingMegAlign™ sequence alignment software using the Clustal algorithm). Theinitial pairwise alignment parameters are set to a K-tuple of 1, a GAPpenalty of 3, a window of 5, and diagonals saved set to =5. The multiplealignment parameters are set at a GAP penalty of 10, and a GAP lengthpenalty of 10.)

NEOKINE

Cytokines are small peptide molecules produced by a variety of cellsthat mediate a wide range of biological activities. Arai et al. (1990)Annu. Rev. Biochem. 59:783 and Paul and Seder (1994) Cell 76:241.Through a complex network, cytokines regulate functions includingcellular growth, inflammation, immunity, differentiation and repair.Mosmann (1991) Curr. Opin. Immunol. 3:311. One superfamily of cytokines,termed the chemokine superfamily, is a large group of more than 30 smallproteins, many of which play a role in the selective recruitment andactivation of leukocytes during inflammation. Wells and Peitsch (1997)J. Leukoc. Biol. 61:5. The chemokine superfamily can be subdivided intotwo groups based on the arrangement of the first two of four conservedcysteines, which are either separated by one amino acid (CXC chemokines)or adjacent (CC chemokines). Baggiolini et al. (1995) Int. J.Immunopharmacol. 17:2. IL-8 and the other CXC chemokines actpreferentially on neutrophils, while the CC chemokines (MCP-1, MCP-2,MCP-3, RANTES, MIP-1 alpha and MIP-1 beta) act on monocytes, but notneutrophils, and have additional activities toward basophil andeosinophil granulocytes, and T-lymphocytes. Baggiolini et al., supra.

The CXC chemokine family of cytokines display disparate angiogenicactivity depending upon the presence or absence of the ELR motif, astructural amino acid motif previously found to be important inreceptor:ligand binding on neutrophils. CXC chemokines containing theELR motif are potent angiogenic factors, inducing both in vitroendothelial chemotaxis and in vivo corneal neovascularization. Incontrast, the CXC chemokines that lack the ELR motif including, PF4,IP-10, and MIG, not only fail to induce significant in vitro endothelialcell chemotaxis or in vivo corneal neovascularization, but are found tobe potent angiostatic factors in the presence of CXC chemokinescontaining the ELR motif. Strieter et al. (1995) Shock 4:3. The CXCcytokines have a signature pattern which spans the region that includesthe four conserved cysteine residues. A CXC-signature pattern has beengenerated from the consensus of multiple CXC chemokines. The signaturepattern has been assigned Prosite Signature PS00471.

The present invention is based on the discovery of family of molecules,referred to herein as NEOKINE protein and nucleic acid molecules. TheNEOKINEs are members of the non-ELR-CXC subfamily of chemokines (ashortening of chemoattractant cytokines). The CXC-chemokines displayfour highly conserved cysteine amino acid residues, with the first twocysteines separated by one non-conserved amino acid residue. The cloningof the NEOKINE family of CXC chemokines revealed at least three atypicalfeatures which distinguish them from previously characterizedchemokines. These are (1) the presence of approximately 5 residuesbetween the third and fourth conserved cysteine residues which areabsent from other CXC chemokines; (2) the fewest residues preceeding thepredicted amino terminus of the mature form of any naturally-occurringchemokine; and (3) a general dissimilarity to all other chemokines inthe region between the second and third conserved cysteines.Phylogenetic analysis of known chemokines (e.g., known CXC as well as CCchemokines) further demonstrates that NEOKINE is unique from otherchemokines subfamilies identified to date although it is clearly arelated chemokine.

The family of NEOKINE chemokines described herein include human, murine,rat and macaque NEOKINE-1. Upon comparison of the sequences for eachspecies orthologue, it was noticed that the NEOKINE-1 chemokinesdisplayed a remarkable degree of identity between orthologues. Forexample, human NEOKINE-1 is 92% identical at the amino acid level tomurine NEOKINE-1 (as determined using a Lipman-Pearson algorithm (Lipmanand Pearson (1985) Science 227:1435-1441, Ktuple=2, Gap Penalty=4, GapLength Penalty=12).

The present invention is also based on the discovery that NEOKINE is thesurrogate ligand for a previously-identified orphan receptor known inthe art as RDC1. RDC1 was first identified as one of four orphanreceptors cloned from a dog thyroid cDNA library based on homology tothe seven-transmembrane helice-containing G-protein coupled receptors(Libert et al. (1989) Science 244:569-572). Three of these, RDC4, RDC7,and RDC8 have since been identified as 5-HT_(1D), adenosine A₁ andadenosine A₂ receptors, respectively (Maenhaut et al. (1991) Biochem.Biophys. Res. Commun. 173:1169-1178; Libert et al. (1991) EMBO J.10:1677-1682; and Maenhaut et al. (1991) Biochem. Biophys. Res. Commun.180:1460-1468). A human orthologue of the fourth orphan, termed GPRN1,was subsequently cloned which was initially proposed to be the receptorfor vasoactive intestinal peptide (“VIP”) (Sreedharan et al. (1991)Proc. Nat'l. Acad. Sci. USA 88:4986-4990). This finding has morerecently been refuted (Nagata et al. (1992) Trends Biochem. Sci.13:102-103; and Cook et al. (1992) FEBS Lett. 300:149-152), leaving thereceptor orphaned. Recent reports have characterized the tissueexpression of RDC1/GPRN1 receptor (Law and Rosenzweig (1994) Biochem.Biophys. Res. Commun. 201:458-465) and have identified and characterizeda murine homologue of RDC1/GPRN1 (Heesen et al. (1998) Immunogenetics47:364-370).

Sequence comparison of human and murine RDC1/GPRN1 reveals that theseorthologues exhibit remarkable sequence identity as is the case withspecies orthologues of NEOKINE. Moreover, phylogenetic analysis ofchemokine receptors demonstrates that RDC1/GPRN1 is a unique butdistantly related member of the CXC subfamily of chemokine receptors.Given the remarkable sequence identity between species orthologues ofNEOKINE and between orthologues of RDC1/GPRN1, as well as the uniquepositions which both NEOKINE and RDC1/GPRN1 fall on their respectivephylogenetic trees, it has been determined that the NEOKINE is thesurrogate ligand for the previously orphaned RDC1/GPRN1 receptor.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally occurring and can be from eitherthe same or different species. For example, a family can contain a firstprotein of human origin, as well as other, distinct proteins of humanorigin or alternatively, can contain homologues of non-human origin.Members of a family may also have common functional characteristics.

In one embodiment, the NEOKINE proteins of the present invention areproteins having an amino acid sequence of about 75-125, preferably about80-120, more preferably about 85-115, more preferably about 90-110, andeven more preferably about 95-105 amino acids containing 3-7, preferably5-6, and more preferably 4 cysteine residues which are conserved betweenfamily members. In another embodiment, a NEOKINE family member isidentified based on the presence of at least one “NEOKINE CXC signaturemotif” in the protein or corresponding nucleic acid molecule. As usedherein, the term “NEOKINE CXC signature motif” refers to a proteindomain having an amino acid sequence of about 35-65, preferably about40-60, more preferably about 45-55 amino acid residues, and even morepreferably at least about 48-50 amino acids containing 3-7, preferably5-6, and more preferably 4 cysteine residues which are conserved betweenfamily members, the first three residues of the NEOKINE CXC signaturemotif having the sequence C-X-C (“CXC”), wherein X is any amino acid andC is cysteine. In a preferred embodiment, a NEOKINE CXC signature motifhas the pattern X (0-2)-C-X-C-X (20-24)-C-X (17-24)-C-X (0-2) (SEQ IDNO:185), wherein X is any amino acid and C is cysteine. In anotherpreferred embodiment, a NEOKINE CXC signature motif has the pattern X(0-2)-C-X-C-X (23, 24)-C-X (20, 21)-C-X (2) (SEQ ID NO:186), wherein Xis any amino acid and C is cysteine. In another preferred embodiment, aNEOKINE CXC signature motif has the pattern X (0-2)-C-X-C-X(6,7)-[LIVMFY]-X (2)-[RKSEQ]-X-[LIVM]-X (2)-[LIVM]-X (8)-C-X (4)-[LIVM](2)-X (13,14)-C-[LIVM]-X (SEQ ID NO:187). In another preferredembodiment, a NEOKINE CXC signature motif has the pattern X (0,1)-[RK]-C-[RK]-C-X(4)-P-X(4,5)-[ED]-X(6)-[KR]-X(5)-C-[DE](2)-X[LIVMFY](4)-X (12,13)-H-C[LIVM]-H (SEQID NO:188). The motifs described herein, are described according tostandard Prosite Signature designation (e.g., X (0-2) designates anyamino acid, n=0-2; X (6, 7) designates any amino acid, n=6 or 7; and[LIVM] indicates any one of Leu, Ile, Val, or Met. All amino acids aredescribed using universal single letter abbreviations according to thesemotifs. In a preferred embodiment, the N-terminal amino acid of theNEOKINE CXC signature motif is the N-terminal amino acid of the matureNEOKINE protein:

Accordingly, in one embodiment, a NEOKINE protein is human NEOKINE-1which contains a NEOKINE CXC signature motif containing about aminoacids 25-72 of SEQ ID NO:116 having the sequence CXC at amino acidresidues 25-27, and having 4 conserved cysteine residues at thepositions indicated in FIG. 8. In another embodiment, a NEOKINE proteinis murine NEOKINE-1 which contains a NEOKINE CXC signature motifcontaining about amino acids 25-72 of SEQ ID NO:119 having the sequenceCXC at amino acid residues 25-27, and having 4 conserved cysteineresidues at the positions indicated in FIG. 8. In another embodiment, aNEOKINE protein is rat NEOKINE-1 which contains a NEOKINE CXC signaturemotif containing at least amino acids 4-51 of SEQ ID NO:122, having 4conserved cysteine residues at the positions indicated in FIG. 8. Inanother embodiment, a NEOKINE protein is macaque NEOKINE-1 whichcontains a NEOKINE CXC signature motif containing at least amino acids20-67 of SEQ ID NO:135, having 4 conserved cysteine residues at thepositions indicated in FIG. 8.

In another embodiment of the invention, a NEOKINE protein has at leastone NEOKINE CXC signature motif and a signal sequence. As used herein, a“signal sequence” refers to a peptide containing about 20 amino acidswhich occurs at the N-terminus of secretory and integral membraneproteins and which contains a majority of hydrophobic amino acidresidues. For example, a signal sequence contains at least about 14-28amino acid residues, preferably about 16-26 amino acid residues, morepreferably about 18-24 amino acid residues, and more preferably about20-22 amino acid residues, and has at least about 40-70%, preferablyabout 50-65%, and more preferably about 55-60% hydrophobic amino acidresidues (e.g., Alanine, Valine, Leucine, Isoleucine, Phenylalanine,Tyrosine, Tryptophan, or Proline). Such a “signal sequence”, alsoreferred to in the art as a “signal peptide”, serves to direct a proteincontaining such a sequence to a lipid bilayer. For example, in oneembodiment, a NEOKINE-1 protein contains a signal sequence of aboutamino acids 1-22 of SEQ ID NO:116. In another embodiment, a NEOKINE-2protein contains a signal sequence of about amino acids 1-22 of SEQ IDNO:119. In another embodiment, a NEOKINE-2 protein contains a signalsequence of about amino acids 1-17 of SEQ ID NO:135.

Accordingly, one embodiment of the invention features a NEOKINE proteinhaving at least a NEOKINE CXC signature motif. Another embodimentfeatures a NEOKINE protein having at least a NEOKINE CXC signature motifand a signal peptide.

Preferred NEOKINE molecules of the present invention have an amino acidsequence sufficiently homologous to the amino acid sequence of SEQ IDNO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125. As used herein,the term “sufficiently homologous” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains have at least about 50% homology,preferably 60% homology, more preferably 70%-80%, and even morepreferably 90-95% homology across the amino acid sequences of thedomains and contain at least one and preferably two structural domains,are defined herein as sufficiently homologous. Furthermore, amino acidor nucleotide sequences which share at least 50%, preferably 60%, morepreferably 70-80, or 90-95% homology and share a common functionalactivity are defined herein as sufficiently homologous.

As used interchangeably herein, an “NEOKINE activity”, “biologicalactivity of NEOKINE” or “functional activity of NEOKINE”, refers to anactivity exerted by a NEOKINE protein, polypeptide or nucleic acidmolecule on a NEOKINE responsive cell as determined in vivo, or invitro, according to standard techniques. In one embodiment, a NEOKINEactivity is a direct activity, such as an association with aNEOKINE-target molecule. As used herein, a “target molecule” or “bindingpartner” is a molecule with which a NEOKINE protein binds or interactsin nature, such that NEOKINE-mediated function is achieved. A NEOKINEtarget molecule can be a non-NEOKINE molecule or a NEOKINE protein orpolypeptide of the present invention. In an exemplary embodiment, aNEOKINE target molecule is a carbohydrate molecule on the cell membrane(e.g., heparan sulfate). In another exemplary embodiment, a NEOKINEtarget molecule is a membrane-bound protein (e.g., a “NEOKINEreceptor”).

In another embodiment, a NEOKINE target is a membrane-bound chemokinereceptor. In another embodiment, a NEOKINE target is an protein molecule(e.g., a “NEOKINE binding partner”). In such an exemplary embodiment, aNEOKINE binding partner can be an non-NEOKINE protein or a secondNEOKINE protein molecule of the present invention. Alternatively, aNEOKINE activity is an indirect activity, such as a cellular signalingactivity mediated by interaction of the NEOKINE protein with a secondprotein (e.g., a NEOKINE receptor or a receptor specific for anotherchemokine).

In one embodiment, a NEOKINE activity is at least one or more of thefollowing activities: (i) interaction of a NEOKINE protein with amembrane-bound NEOKINE receptor (e.g., RDC1); (ii) interaction of aNEOKINE protein with a membrane-bound chemokine receptor; (iii) indirectinteraction of a NEOKINE protein with an intracellular protein via amembrane-bound NEOKINE receptor (e.g., RDC1); (iv) indirect interactionof a NEOKINE protein with an intracellular protein via a membrane-boundchemokine receptor; (v) complex formation between a soluble NEOKINEprotein and a NEOKINE binding partner; (vi) inhibition of theinteraction of chemokines (e.g., pro-inflammatory chemokines) by bindingto their cognate receptors; (vii) inhibition of the binding of HIV toHIV co-receptors; and (vii) inhibition of the binding of HIV to HIVco-receptors wherein said binding induces subsequent infection ofsusceptible cells.

In another embodiment, a NEOKINE activity is at least one or more of thefollowing activities: (1) modulation of cellular signal transduction,either in vitro or in vivo; (2) regulation of gene transcription in acell expressing a NEOKINE receptor or a chemokine receptor; (3)regulation of gene transcription, in a cell expressing a NEOKINEreceptor (e.g., RDC1) or a chemokine receptor, wherein said cell isinvolved in angiogenesis or inflammation; (4) regulation ofangiogenesis; (5) regulation of angiogenesis, wherein said regulationcomprises inhibibition of angiogenesis; (6) regulation of angiogenesis,wherein said regulation comprises maintenance of angiostasis; (7)regulation of inflammation; (8) inhibition of chemoattraction (e.g.,neutrophil chemoattraction); and (9) inhibition of pro-inflammatorychemokines by binding to their cognate receptors.

In a preferred embodiment of the invention a NEOKINE or NEOKINEmodulator is useful for regulating, preventing and/or treating at leastone or more of the following proliferative diseases or disorders: (1)cancers of the epithelia (e.g., carcinomas of the pancreas, stomach,liver, secretory glands (e.g., adenocarcinoma) bladder, lung, breast,skin (e.g., malignant melanoma), reproductive tract including prostategland, ovary, cervix and uterus); (2) cancers of the hematopoietic andimmune system (e.g., leukemias and lymphomas); (3) cancers of thecentral nervous, brain system and eye (e.g., gliomas, neuroblastoma andretinoblastoma); and (4) cancers of connective tissues, bone, musclesand vasculature (e.g., sarcomas).

In yet another embodiment of the invention, a NEOKINE or NEOKINEmodulator is useful for regulating, preventing and/or treating at leastone or more of the following diseases or disorders: (1) inflammation;(2) psoriasis; (3) immune rejection following skin graft; (4) immunerejection following kidney transplant; (5) kidney inflammation in acuterenal failure; (6) brain inflammation following stroke or ischaemia; and(7) brain inflammation following viral infection.

Accordingly, another embodiment of the invention features isolatedNEOKINE proteins and polypeptides having a NEOKINE activity. PreferredNEOKINE proteins have at least one NEOKINE CXC signature motif and aNEOKINE activity. In another preferred embodiment, a NEOKINE proteinfurther comprises a signal sequence. In still another preferredembodiment, a NEOKINE protein has a NEOKINE CXC signature motif, aNEOKINE activity, and an amino acid sequence sufficiently homologous toan amino acid sequence of SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122,or SEQ ID NO:125.

The human NEOKINE-1 cDNA, which is approximately 1564 nucleotides inlength, encodes a protein which is approximately 99 amino acid residuesin length. The human NEOKINE-1 protein contains at least a NEOKINE CXCsignature motif. A NEOKINE CXC signature motif can be found at least,for example, from about amino acids 25-72 of SEQ ID NO:116. The humanNEOKINE-1 protein is predicted to be a secreted protein which furthercontains a signal sequence at about amino acids 1-22 of SEQ ID NO:116.The prediction of such a signal peptide can be made, for example,utilizing the computer algorithm SIGNALP (Henrik, et al. (1997) ProteinEngineering 10:1-6).

The murine NEOKINE-1 cDNA, which is approximately 1564 nucleotides inlength, encodes approximately 99 amino acid residues of the murineNEOKINE-1 protein. The murine NEOKINE-1 protein contains a NEOKINE CXCsignature motif. A NEOKINE CXC signature motif can be found at least,for example, from about amino acids 25-72 of SEQ ID NO:119. The murineNEOKINE-1 protein is predicted to be a secreted protein which furthercontains a signal sequence at about amino acids 1-22 of SEQ ID NO:119.

The rat NEOKINE-1 cDNA, which is approximately 1372 nucleotides inlength, encodes approximately 79 amino acid residues of the ratNEOKINE-1 protein. The rat NEOKINE-1 protein contains a NEOKINE CXCsignature motif. A NEOKINE CXC signature motif comprises at least aboutamino acids 4-51 of SEQ ID NO:122. The rat NEOKINE-1 protein ispredicted to be a secreted protein.

The macaque NEOKINE-1 cDNA, which is approximately 1458 nucleotides,encodes approximately 94 amino acid residues of the macaque NEOKINE-1protein. The macaque NEOKINE-1 protein contains a NEOKINE CXC signaturemotif. A NEOKINE CXC signature motif comprises at least about aminoacids 20-67 of SEQ ID NO:135. The macaque NEOKINE-1 protein is predictedto be a secreted protein which further contains a signal sequenceincluding at least amino acids 1-17 of SEQ ID NO:135.

TANGO 129

Members of the tumor necrosis factor (TNF) superfamily and theirreceptors, both of which are expressed on activated T cells andelsewhere, are thought to play an important role in T-cell activationand stimulation, cell proliferation and differentiation, as well asapoptosis.

Proteins that are members of the TNF superfamily initiate signaltransduction by binding to receptors, members of the TNF receptor (TNFR)superfamily, which lack intrinsic catalytic activity. This is in markedcontrast epidermal growth factor and platelet-derived growth factor bothof which bind to receptors having an intracellular tyrosine kinasedomain which causes receptor autophosphorylation and initiatesdownstream phosphorylation events.

Members of the TNFR superfamily carry out signal transduction byinteracting with members of the Janus or JAK family of tyrosine kinases.In turn, JAK family members interact with STAT (signal transducers andactivators of transcription) family members, a class of transcriptionalactivators.

The present invention is based on the discovery of a cDNA moleculeencoding human TANGO129 (T129), a member of the TNF receptorsuperfamily.

A nucleotide sequence encoding a human T129 protein is shown in SEQ IDNO:137; and SEQ ID NO:139 (open reading frame only). A predicted aminoacid sequence of T129 protein is also shown in SEQ ID NO:138.

The T129 cDNA of SEQ ID NO:137, which is approximately 2570 nucleotideslong including untranslated regions, encodes a protein amino acid havinga molecular weight of approximately 46 kDa (excluding post-translationalmodifications).

Alignment of the TNFR/NGFR cysteine-rich domain of human T129 protein(SEQ ID NO:142) with a TNFR/NGFR cysteine-rich domain consensus derivedfrom a hidden Markov model (PF00020; SEQ ID NO:5), revealed somesimilarity.

An approximately 3.0 kb T129 mRNA transcript is expressed at a moderatelevel in peripheral blood leukocytes, spleen, and skeletal muscle. Lowerlevels of this transcript were observed in heart, brain, and placenta.Human T129 is one member of a family of molecules (the “T129 family”)having certain conserved structural and functional features. The term“family” when referring to the protein and nucleic acid molecules of theinvention is intended to mean two or more proteins or nucleic acidmolecules having a common structural domain and having sufficient aminoacid or nucleotide sequence identity as defined herein. Such familymembers can be naturally occurring and can be from either the same ordifferent species. For example, a family can contain a first protein ofhuman origin and a homologue of that protein of murine origin, as wellas a second, distinct protein of human origin and a murine homologue ofthat protein. Members of a family may also have common functionalcharacteristics.

In one embodiment, a T129 protein includes a TNFR/NGFR domain having atleast about 65%, preferably at least about 75%, and more preferablyabout 85%, 95%, or 98% amino acid sequence identity to the TNFR/NGFRdomain of SEQ ID NO:141.

Preferred T129 polypeptides of the present invention have an amino acidsequence sufficiently identical to the TNFR/NGFR domain amino acidsequence of SEQ ID NO:141. As used herein, the term “sufficientlyidentical” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences have acommon structural domain and/or common functional activity. For example,amino acid or nucleotide sequences which contain a common structuraldomain having about 65% identity, preferably 75% identity, morepreferably 85%, 95%, or 98% identity are defined herein as sufficientlyidentical.

As used interchangeably herein a “T129 activity”, “biological activityof T129” or “functional activity of T129”, refers to an activity exertedby a T129 protein, polypeptide or nucleic acid molecule on a T129responsive cell as determined in vivo, or in vitro, according tostandard techniques. A T129 activity can be a direct activity, such asan association with or an enzymatic activity on a second protein or anindirect activity, such as a cellular signaling activity mediated byinteraction of the T129 protein with a second protein. In a preferredembodiment, a T129 activity includes at least one or more of thefollowing activities: (i) interaction with proteins in the T129signaling pathway (ii) interaction with a T129 ligand; or (iii)interaction with an intracellular target protein.

Accordingly, another embodiment of the invention features isolated T129proteins and polypeptides having a T129 activity.

Yet another embodiment of the invention features T129 molecules whichcontain a signal sequence. Generally, a signal sequence (or signalpeptide) is a peptide containing about 20 amino acids which occurs atthe extreme N-terminal end of secretory and integral membrane proteinsand which contains large numbers of hydrophobic amino acid residues andserves to direct a protein containing such a sequence to a lipidbilayer.

A259

The A259 proteins and nucleic acid molecules comprise a family ofmolecules having certain conserved structural and functional features.As used herein, the term “family” is intended to mean two or moreproteins or nucleic acid molecules having a common structural domain andhaving sufficient amino acid or nucleotide sequence identity as definedherein. Family members can be from either the same or different species.For example, a family can comprises two or more proteins of humanorigin, or can comprise one or more proteins of human origin and one ormore of non-human origin. Members of the same family may also havecommon structural domains.

For example, A259 proteins of the invention have signal sequences. Asused herein, a “signal sequence” includes a peptide of at least about 15or 20 amino acid residues in length which occurs at the N-terminus ofsecretory and membrane-bound proteins and which contains at least about70% hydrophobic amino acid residues such as alanine, leucine,isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. Ina preferred embodiment, a signal sequence contains at least about 10 to40 amino acid residues, preferably about 15-30 amino acid residues, morepreferably about 22 amino acid residues, and has at least about 60-80%,more preferably 65-75%, and more preferably at least about 70%hydrophobic residues. A signal sequence serves to direct a proteincontaining such a sequence to a lipid bilayer. Thus, in one embodiment,an A259 protein contains a signal sequence at about amino acids 1 to 22of SEQ ID NO:147 (SEQ ID NO:149). The signal sequence is cleaved duringprocessing of the mature protein.

In another example, an A259 family member also includes one or more ofthe following domains: (1) an extracellular domain; (2) a transmembranedomain; and (3) a cytoplasmic domain. Thus, in one embodiment, an A259protein contains an extracellular domain at about amino acids 1 to 1141of SEQ ID NO:147 (SEQ ID NO:150). In another embodiment, an A259 proteincontains a transmembrane domain at about amino acids 1142 to 1164 of SEQID NO:147 (SEQ ID NO:159). In another embodiment, an A259 proteincontains a cytoplasmic domain at about amino acids 1165 to 1188 of SEQID NO:147 (SEQ ID NO:160).

In one embodiment, the extracellular domain of A259 can include aninserted domain (“I” domain). In another embodiment, the I domain islocated between integrin α-subunit repeat domains 2 and 3.

In a preferred embodiment, an A259 family member has the amino acidsequence of SEQ ID NO:147 wherein the extracellular domain is located atabout amino acids 1 to 1141 (SEQ ID NO:150), the I domain (SEQ IDNO:151) is located at about amino acid positions 164 to 345, and theintegrin α-subunit repeat domains on either side of the I domain(integrin α-subunit repeat domains 2 and 3) are located at amino acidpositions 115 to 157 (SEQ ID NO:153) and 367 to 392 (SEQ ID NO:154),respectively.

An A259 family member can include one or more integrin α-subunit repeatdomains within the extracellular domain. As used herein, a “repeatdomain” refers to one or more of seven homologous protein domains thatare conserved in integrin α-subunit family members, which are about 10to 65 residues, preferably about 20 to 50 residues, more preferablyabout 25 to 45 amino acid residues, and most preferably about 26 to 43residues, in length, and which are about 10 to 50, preferably about 15to 40, more preferably about 20 to 35 amino acids apart from oneanother.

A repeat domain typically has one of the two following consensussequences. The first repeat consensus sequence (type 1) is:F-G-Xaa(n)-[G or A]-A-[P or L or Q] (SEQ ID NO:189), wherein F isphenylalanine, G is glycine, Xaa is any amino acid, n is about 10 to 30amino acid residues, preferably about 15 to 20 amino acid residues, morepreferably about 16 to 19 amino acid residues, A is alanine, P isproline, L is leucine, and Q is glutamine. The second repeat consensussequence (type 2) is: [G or S]-[F or Y]-Xaa(n)-[G or V or L]-[A or M]-[Por Y] (SEQ ID NO:190), wherein G is glycine, S is serine, F isphenylalanine, Y is tyrosine, Xaa is any amino acid, n is about 5 to 40amino acid residues, preferably about 10 to 35 amino acid residues, morepreferably about 14 to 33 amino acid residues, V is valine, A isalanine, M is methionine, and P is proline.

In one embodiment, an A259 family member includes one or more repeatdomains having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 39 to 74, and/or 115 to 157, and/or 367 to392, and/or 421 to 455, and/or 478 to 516, and/or 540 to 575, and/or 602to 640 of SEQ ID NO:147, which are the repeat domains of A259 (theserepeat domains are also represented as SEQ ID NO:152, 153, 154, 155,156, 157, and 158, respectively). In another embodiment, an A259 familymember includes one or more repeat domains having an amino acid sequencethat is at least about 55%, preferably at least about 65%, morepreferably at least about 75%, yet more preferably at least about 85%,and most preferably at least about 95% identical to amino acids 39 to74, and/or 115 to 157, and/or 367 to 392, and/or 421 to 455, and/or 478to 516, and/or 540 to 575, and/or 602 to 640 of SEQ ID NO:147, which arethe repeat domains of A259 (these repeat domains are also represented asSEQ ID NO:152, 153, 154, 155, 156, 157, and 158, respectively), and hasone or more repeat consensus sequences described herein. In anotherembodiment, an A259 family member includes one or more repeat domainshaving an amino acid sequence that is at least about 55%, preferably atleast about 65%, more preferably at least about 75%, yet more preferablyat least about 85%, and most preferably at least about 95% identical toamino acids 39 to 74, and/or 115 to 157, and/or 367 to 392, and/or 421to 455, and/or 478 to 516, and/or 540 to 575, and/or 602 to 640 of SEQID NO:147, which are the repeat domains of A259 (these repeat domainsare also represented as SEQ ID NO:152, 153, 154, 155, 156, 157, and 158,respectively), has one or more I consensus sequences described herein,and has at least one A259 biological activity as described herein.

An A259 family member can also include an inserted, or I domain (alsocalled von Willebrand factor type A domain). As used herein, an “Idomain” refers to a domain that appears in only some of the integrin αsubunits, e.g., α1, α2, αM, and αX, and that is inserted between thesecond and third integrin α-subunit repeat domains. I domains preventthe occurrence of proteolytic cleavage that separates integrin αsubunits into heavy and light fragments that are disulfide linked. An Idomain includes about 100 to 300 amino acid residues, preferably about150 to 200 amino acid residues, more preferably about 160 to 190 aminoacid residues, and most preferably about 182 amino acid residues.

An I domain typically has the following consensus sequence:D-G-S-Xaa-S-Xaa(n1)-F-Xaa(n2)-Q-Y (SEQ ID NO:191), wherein D is asparticacid, G is glycine, S is serine, Xaa is any amino acid, n1 is about 5 to15, preferably about 8 to 12, more preferably about 9 to 11 amino acidresidues, F is phenylalanine, n2 is about 15 to 30, preferably 18 to 28,more preferably about 20 to 26 amino acid residues, Q is glutamine, andY is tyrosine.

In one embodiment, an A259 family member includes an I domain having anamino acid sequence that is at least about 55%, preferably at leastabout 65%, more preferably at least about 75%, yet more preferably atleast about 85%, and most preferably at least about 95% identical toamino acids 164 to 345 of SEQ ID NO:147, which is the I domain of A259(this I domain is also represented as SEQ ID NO:151). In anotherembodiment, an A259 family member includes an I domain having an aminoacid sequence that is at least about 55%, preferably at least about 65%,more preferably at least about 75%, yet more preferably at least about85%, and most preferably at least about 95% identical to amino acids 164to 345 of SEQ ID NO:147, which is the I domain of A259 (this I domain isalso represented as SEQ ID NO:151), and an I domain consensus sequencedescribed herein. In another embodiment, an A259 family member includesan I domain having an amino acid sequence that is at least about 55%,preferably at least about 65%, more preferably at least about 75%, yetmore preferably at least about 85%, and most preferably at least about95% identical to amino acids 164 to 345 of SEQ ID NO:147, which is the Idomain of A259 (this I domain is also represented as SEQ ID NO:151). Inanother embodiment, an A259 family member includes an I domain having anamino acid sequence that is at least about 55%, preferably at leastabout 65%, more preferably at least about 75%, yet more preferably atleast about 85%, and most preferably at least about 95% identical toamino acids 164 to 345 of SEQ ID NO:147, which is the I domain of A259(this I domain is also represented as SEQ ID NO:151), has one or more Idomain consensus sequences described herein, and has at least one A259biological activity as described herein.

An A259 family member can also include a cytoplasmic domain. Thecytoplasmic domain typically includes about 10 to 40, preferably about20 to 30, more preferably about 22 to 28, still more preferably about 22to 26 amino acid residues in length. The A259 cytoplasmic domaintypically has the following consensus sequence: [F or Y or W or S]-[R orK]-Xaa(n1)-G-F-F-Xaa(n2)-R (SEQ ID NO:192), wherein F is phenylalanine,Y is tyrosine, W is tryptophan, S is serine, R is arginine, K is lysine,Xaa is any amino acid, n1 and n2 are 0 to 1, and G is glycine.

In a preferred embodiment, an A259 family member has the amino acidsequence of SEQ ID NO:147 wherein the cytoplasmic domain is located atabout amino acids 1165 to 1188 (SEQ ID NO:160) and the cytoplasmicdomain consensus sequence is located from about amino acid 1165 to 1170.

Various features of human and murine A259 are summarized below.

Human A259

A cDNA encoding human A259 was identified by analyzing the sequences ofclones present in an LPS stimulated human primary osteoblast cDNAlibrary. This analysis led to the identification of a clone, Atho002i17,encoding full-length human A259. The human A259 cDNA of this clone is5042 nucleotides long (SEQ ID NO:145). The open reading frame of thiscDNA, nucleotides 127 to 3690 of SEQ ID NO:145 (SEQ ID NO:146), encodesa 1188 amino acid receptor protein (SEQ ID NO:147).

The signal peptide prediction program SIGNALP (Nielsen et al. (1997)Protein Engineering 10:1-6) predicted that human A259 includes a 22amino acid signal peptide (amino acid 1 to about amino acid 22 of SEQ IDNO:147; SEQ ID NO:149) preceding the mature A259 protein (correspondingto about amino acid 23 to amino acid 1188 of SEQ ID NO:147) (SEQ IDNO:148). The human A259 protein molecular weight is 133.5 kDa prior tothe cleavage of the signal peptide, 131.1 kDa after cleavage of thesignal peptide.

Human A259 includes a extracellular domain (about amino acids 1 to 1141of SEQ ID NO:147; SEQ ID NO:150), an I domain (also called a VonWillebrand Factor type A domain; about amino acids 164 to 345 of SEQ IDNO:147; SEQ ID NO:151), seven repeat domains (about amino acids 39 to74, 115 to 157, 367 to 392, 421 to 455, 478 to 516, 540 to 575, and 602to 640 of SEQ ID NO:147 (SEQ ID NO:152, 153, 154, 155, 156, 157, and158, respectively)), a transmembrane domain (about amino acids 1142 to1164 of SEQ ID NO:147; SEQ ID NO:159), and a cytoplasmic domain (aboutamino acids 1165 to 1188 of SEQ ID NO:147; SEQ ID NO:160).

A slightly different model of the integrin alpha repeat domainidentifies five integrin alpha repeat domains within human A259 (aboutamino acids 37-90, 421-472, 476-532, 538-593, and 600-654 of SEQ IDNO:147 (SEQ ID NOS:179, 180, 181, 182, and 183, respectively)). Thesedomains were identified by homology searching using consensus domainsderived from hidden Markov models (HMMs). HMMs can be used to domultiple sequence alignment and very sensitive database searching, usingstatistical descriptions of a sequence family's consensus. FIG. 12Adepicts an alignment of amino acids 37-90 of human A259 (SEQ ID NO:179)with a consensus integrin alpha repeat domain derived from a hiddenMarkov model (SEQ ID NO:184). FIG. 12B depicts an alignment of aminoacids 421-472 of human A259 (SEQ ID NO:180) with a consensus integrinalpha repeat domain derived from a hidden Markov model (SEQ ID NO:184).FIG. 12C depicts an alignment of amino acids 476-532 of human A259 (SEQID NO:181) with a consensus integrin alpha repeat domain derived from ahidden Markov model (SEQ ID NO:184). FIG. 12D depicts an alignment ofamino acids 538-593 of human A259 (SEQ ID NO:182) with a consensusintegrin alpha repeat domain derived from a hidden Markov model (SEQ IDNO:184). FIG. 12E depicts an alignment of amino acids 600-654 of humanA259 (SEQ ID NO:183) with a consensus integrin alpha repeat domainderived from a hidden Markov model (SEQ ID NO:184).

Predicted N-glycosylation sites are found from amino acids 82 to 85, 95to 98, 291 to 294, 331 to 334, 358 to 361, 449 to 452, 462 to 465, 528to 531, 642 to 645, 694 to 697, 857 to 860, 894 to 897, 973 to 976, 1031to 1034, 1039 to 1042 and 1059 to 1062 of SEQ ID NO:147. A cAMP andcGMP-dependent protein kinase phosphorylation site is found from aminoacids 700 to 703 of SEQ ID NO:147.

Predicted protein kinase C phosphorylation sites are found from aminoacids 27 to 29, 97 to 99, 221 to 223, 287 to 289, 355 to 357, 434 to436, 451 to 453, 530 to 532, 548 to 550, 587 to 589, 662 to 664, 703 to705, 716 to 718, 770 to 772, 833 to 835, 938 to 940, 1092 to 1094, 1100to 1102, 1171 to 1173 and 1183 to 1185 of SEQ ID NO:147.

Predicted casein kinase II phosphorylation sites are found from aminoacids 161 to 164, 221 to 224, 230 to 233, 270 to 273, 287 to 290, 322 to325, 485 to 488, 530 to 533, 548 to 551, 556 to 559, 593 to 596, 662 to665, 696 to 669, 724 to 727, 753 to 756, 801 to 804, 938 to 941, 966 to969, 974 to 977, 1046 to 1049, 1061 to 1064 and 1133 to 1136 of SEQ IDNO:147.

A predicted tyrosine kinase phosphorylation site is found from aminoacids 812 to 820 of SEQ ID NO:147.

Predicted N-myristoylation sites are found from amino acids 6 to 11, 35to 40, 106 to 111, 236 to 241, 245 to 250, 257 to 262, 354 to 359, 363to 368, 379 to 384, 422 to 427, 473 to 478, 481 to 486, 543 to 548, 625to 630, 690 to 695, 725 to 730, 877 to 882, 930 to 935 and 1147 to 1152of SEQ ID NO:147.

A predicted amidation site is found from amino acids 257 to 260 of SEQID NO:147. An immunoglobulin and major histocompatibility complexproteins signature is found from amino acids 74 to 80 of SEQ ID NO:147.

Human A259 is homologous to human integrin α10 (SEQ ID NO:161; GenBankAccession Number AF074015), a β1-associated collagen binding integrin(Camper, Hellman, and Lundgren-Akerlund (1998) Journal of Biol. Chem.273, 32:20383-20389). The human A259 signal sequence is represented byamino acids 1 to 22 (and encoded by nucleotides 127 to 192 of SEQ IDNO:145). The human α10 integrin signal sequence is represented by aminoacids 1 to 22 (and encoded by nucleotides 22 to 87 of SEQ ID NO:161).The human A259 extracellular domain sequence (SEQ ID NO:150) isrepresented by amino acids 1 to 1141 (and encoded by nucleotides 127 to3549 of SEQ ID NO:145), and the human α10 extracellular domain sequenceis represented by amino acids 1 to 1098 (and encoded by nucleotides 22to 3381 of SEQ ID NO:161). The human A259 I domain (SEQ ID NO:151) isrepresented by amino acids 164 to 345 (and encoded by nucleotides 616 to1161 of SEQ ID NO:145), and the human α10 I domain is represented byamino acids 140 to 337 (and encoded by nucleotides 505 to 1098 of SEQ IDNO:161). The human A259 transmembrane domain (SEQ ID NO:159) isrepresented by amino acids 1142 to 1164 (and encoded by nucleotides 3550to 3618 of SEQ ID NO:145), and the human α10 transmembrane domain isrepresented by amino acids 1099 to 1123 (and encoded by nucleotides 3382to 3456 of SEQ ID NO:161). The human A259 cytoplasmic domain (SEQ IDNO:160) is represented by amino acids 1165 to 1188 (and encoded bynucleotides 3619 to 3690 of SEQ ID NO:145), and the human α10cytoplasmic domain is represented by amino acids 1124 to 1145 (andencoded by nucleotides 3457 to 3522 of SEQ ID NO:161).

Alignments have shown that there is an overall 46.3% identity betweenthe full length human A259 nucleic acid molecule and the full lengthhuman α10 molecule, and a 43.0% identity between the A259 amino acidsequence and the human α10 amino acid sequence. There is also a 55.1%identity between the human A259 and the human α10 open reading frames.There is also an overall 37.6% amino acid identity, and a 41.5% fulllength nucleic acid identity, between the full length human A259 nucleicacid molecule and the full length human α1 (VLA-1 (very late antigen-1)molecule.

Human A259 is homologous to murine A259. The human A259 signal sequenceis represented by amino acids 1 to 22 (and encoded by nucleotides 127 to192 of SEQ ID NO:145). The murine A259 signal sequence is represented byamino acids 1 to 22 (and encoded by nucleotides 28 to 93 of SEQ IDNO:163). The human A259 extracellular domain sequence (SEQ ID NO:150) isrepresented by amino acids 1 to 1141 (and encoded by nucleotides 127 to3549 of SEQ ID NO:145), and the murine A259 extracellular domainsequence is represented by amino acids 1 to 1141 (and encoded bynucleotides 28 to 3450 of SEQ ID NO:163). The human A259 I domain (SEQID NO:151) is represented by amino acids 164 to 345 (and encoded bynucleotides 616 to 1161 of SEQ ID NO:145), and the murine A259 I domainis represented by amino acids 164 to 345 (and encoded by nucleotides 517to 1062 of SEQ ID NO:163). The human A259 repeat domains are representedby amino acids 39 to 74, 115 to 157, 367 to 392, 421 to 455, 478 to 516,540 to 575, and 602 to 640 (and encoded by nucleotides 244 to 348, 469to 597, 1225 to 1302, 1387 to 1491, 1558 to 1674, 1744 to 1851, and 1930to 2046, respectively, of SEQ ID NO:145), and the murine A259 repeatdomains are represented by amino acids 39 to 74, 115 to 157, 367 to 392,421 to 455, 478 to 516, 540 to 575, and 602 to 640 (and encoded bynucleotides 145 to 249, 370 to 498, 1126 to 1203, 1288 to 1392, 1459 to1575, 1645 to 1752, and 1831 to 1947, respectively, of SEQ ID NO:163).The human A259 transmembrane domain (SEQ ID NO:159) is represented byamino acids 1142 to 1164 (and encoded by nucleotides 3550 to 3618 of SEQID NO:145), and the murine A259 transmembrane domain is represented byamino acids 1142 to 1164 (and encoded by nucleotides 3283 to 3357 of SEQID NO:163). The human A259 cytoplasmic domain (SEQ ID NO:160) isrepresented by amino acids 1165 to 1188 (and encoded by nucleotides 3619to 3690 of SEQ ID NO:145), and the murine A259 cytoplasmic domain isrepresented by amino acids 1165 to 1188 (and encoded by nucleotides 3358to 3423 of SEQ ID NO:163).

Alignments show that there is an overall 78.4% identity between the fulllength human A259 nucleic acid molecule and the full length murine A259molecule, and a 90.3% identity between the A259 amino acid sequence andthe murine A259 amino acid sequence. There is also an 86.8% identitybetween the human and murine A259 open reading frames.

Clone Human I2, which encodes human A259, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on Apr. 2, 1999 and assigned Accession Number 207190.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. 112.

FIG. 10 depicts a hydropathy plot of human A259. Relatively hydrophobicregions of the protein are shown above the horizontal line, andrelatively hydrophilic regions of the protein are below the horizontalline. The cysteine residues (cys) and predicted N-glycosylation sitesare indicated by short vertical lines just below the hydropathy trace.The dashed vertical line separates the signal sequence (amino acids 1 to22 of SEQ ID NO:147; SEQ ID NO:149) on the left from the mature protein(amino acids 23 to 1188 of SEQ ID NO:147; SEQ ID NO:148) on the right.The A259 transmembrane domain is indicated by the section of the plotunder which the number 6.4 can be seen, which represents a scoreassigned to the predicted transmembrane domain. The extracellular domain(SEQ ID NO:150) and cytoplasmic domain (SEQ ID NO:160) are similarlyindicated by gray horizontal bars, labeled as “out” and “in”,respectively.

An electronic survey of proprietary databases revealed that A259 isfound in abundantly in human and mouse osteoblast cDNA libraries andalso in human bone marrow and neuronal cDNA libraries. In addition, in aNorthern blot containing RNA from human tissues, A259 was expressed astwo messages, a 6 kb and a 4 kb message, in RNA from human osteoblasts.The sequence used to probe this human Northern blot contained human A259C-terminal and 3′ untranslated regions. The expression level in humanosteoblasts did not change when stimulated with TNF-α.

The expression of human A259 was measured in various clinical liversamples taken from subjects not suffering from liver fibrosis and frompatients suffering from liver fibrosis using TaqMan quantitative PCR.The results of this analysis are depicted in FIG. 13A. Human A259 wasexpressed at a lower level in the livers of patients not suffering fromliver fibrosis (A, B, and C; relative expression 0.13, 0.29, and 0.09,respectively) than in the livers of patients suffering from liverfibrosis (D, E, F, G, H, I, J, and K; relative expression 1.59, 0.50,0.55, 1.56, 0.62, 0.93, 3.54, and 0.61, respectively).

The expression of human A259 was measured in various cells using TaqManquantitative PCR. The results of this analysis are depicted in FIG. 13B.The cells tested were: heart (A; relative expression 1.69), lung (B;relative expression 0.41), liver (C; relative expression 0.10), passagedstellate cells (D; relative expression 36.15), quiescent stellate cells(E; relative expression 0.15), stellate cells (F; relative expression18.71); stellate/F BS cells (G; relative expression 31.03), NHDFfibroblasts (H; relative expression 0.71); TGF-treated NHDF fibroblasts(I; relative expression 12.69); NHLF fibroblasts (J; relative expression4.81); and TGF-treated NHLF fibroblasts (K; relative expression 66.06).

Human A259 maps to human chromosome location hu15q22. The flankingmarkers are AFM094YC1 and NIB1778. The nearby known disease lociinclude: CLN6 (neuronal ceroid-lipofuscinosis) and BBS4 (Bardet-BiedlSyndrome 4). The nearby known genes include: HDC (histidinedecarboxylase), LIPC (hepatic lipase), PKM2 (pyruvate kinase 3), CLN6(neuronal ceroid-lipofuscinosis), BBS4 (Bardet-Biedl Syndrome 4), andNMB (neuromedin B). The murine syntenic chromosomal location is mo9. Theknown nearby loci include: rc (rough coat), ash (ashen), flail(flailer), pml (promyelotic leukemia), and sty (small thymus). The knownnearby genes include: hpl (hepatitic lipase).

Murine A259

A cDNA encoding human A259 was identified by analyzing the sequences ofclones present in a murine bone marrow cDNA library. This analysis ledto the identification of a clone, AtmMa113d1, encoding full-lengthmurine A259. The murine A259 cDNA of this clone is 4858 nucleotides long(SEQ ID NO:163). The open reading frame of this cDNA, nucleotides 28 to3591 of SEQ ID NO:163 (SEQ ID NO:164), encodes a 1188 amino acidreceptor protein (SEQ ID NO:165).

The signal peptide prediction program SIGNALP (Nielsen et al. (1997)Protein Engineering 10:1-6) predicted that murine A259 includes a 22amino acid signal peptide (amino acid 1 to about amino acid 22 of SEQ IDNO:165) (SEQ ID NO:167) preceding the mature A259 protein (correspondingto about amino acid 23 to amino acid 1188 of SEQ ID NO:165) (SEQ IDNO:166). The mouse A259 protein molecular weight is 133.1 kDa prior tothe cleavage of the signal peptide, 130.5 kDa after cleavage of thesignal peptide.

Murine A259 includes a extracellular domain (about amino acids 1 to 1141of SEQ ID NO:165; SEQ ID NO:168), an I domain (about amino acids 164 to345 of SEQ ID NO:165; SEQ 169), seven repeat domains (about amino acids39 to 74, 115 to 157, 367 to 392, 421 to 455, 478 to 516, 540 to 575,and 602 to 640 of SEQ ID NO:165 (SEQ ID NO:170, 171, 172, 173, 174, 175,and 176, respectively)), a transmembrane domain (about amino acids 1142to 1164 of SEQ ID NO:165; SEQ ID NO:177), and a cytoplasmic domain(about amino acids 1165 to 1188 of SEQ ID NO:165; SEQ ID NO:178).

Predicted N-glycosylation sites are found from amino acids 82 to 85, 95to 98, 291 to 294, 331 to 334, 358 to 361, 449 to 452, 462 to 465, 528to 531, 642 to 645, 694 to 697, 857 to 860, 894 to 897, 973 to 976, 1031to 1034, 1039 to 1042 and 1059 to 1062 of SEQ ID NO:165.

Predicted protein kinase C phosphorylation sites are found from aminoacids 51 to 53, 97 to 99, 221 to 223, 287 to 289, 355 to 357, 434 to436, 451 to 453, 530 to 532, 569 to 571, 716 to 718, 770 to 772, 833 to835, 1092 to 1094, 1100 to 1102 and 1171 to 1173 of SEQ ID NO:165.

Predicted casein kinase II phosphorylation sites are found from aminoacids 161 to 164, 221 to 224, 230 to 233, 270 to 273, 287 to 290, 322 to325, 485 to 488, 530 to 533, 548 to 551, 556 to 559, 593 to 596, 696 to669, 724 to 727, 753 to 756, 801 to 804, 859 to 862, 938 to 941, 966 to969, 974 to 977, 1046 to 1049, 1061 to 1064 and 1133 to 1136 of SEQ IDNO:165.

A predicted tyrosine kinase phosphorylation site is found from aminoacids 812 to 820.

Predicted N-myristoylation sites are found from amino acids 6 to 11, 106to 111, 236 to 241, 245 to 250, 257 to 262, 354 to 359, 363 to 368, 379to 384, 477 to 482, 543 to 548, 625 to 630, 690 to 695, 725 to 730, 1030to 1035 and 1147 to 1152 of SEQ ID NO:165.

Predicted amidation site are found from amino acids 51 to 54 and 257 to260 of SEQ ID NO:165.

Clone Mouse 12, which encodes murine A259, was deposited with theAmerican Type Culture Collection (10801 University Boulevard, Manassas,Va. 20110-2209) on Apr. 2, 1999 and assigned Accession Number 207191.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. 112.

FIG. 11 depicts a hydropathy plot of murine A259. Relatively hydrophobicregions of the protein are shown above the horizontal line, andrelatively hydrophilic regions of the protein are below the horizontalline. The cysteine residues (cys) and predicted N-glycosylation sitesare indicated by short vertical lines just below the hydropathy trace.The dashed vertical line separates the signal sequence (amino acids 1 to22 of SEQ ID NO:165; SEQ ID NO:167) on the left from the mature protein(amino acids 23 to 1188 of SEQ ID NO:165; SEQ ID NO:166) on the right.The A259 transmembrane domain is indicated by the section of the plotunder which the number 6.4 can be seen, which represents a scoreassigned to the predicted transmembrane domain. The extracellular domain(SEQ ID NO:168) and cytoplasmic domain (SEQ ID NO:178) are similarlyindicated by gray horizontal bars, labeled as “out” and “in”,respectively.

In situ data of A259 expression in mouse is summarized as follows:During embryogenesis, E13.5 through postnatal day 1.5 tested, alldeveloping bone structures have strong expression. The signal is locatedmostly at the edge of the developing bones. Beginning at E14.5 the bonemarrow space can be seen and is negative for expression. However, somelarge bones have signal in the bone, not just at the edge. Signal isalso observed in the small intestine, diaphragm and to a lesser extentthe muscle layer under the skin. The diaphragm and intestine expressionpattern is suggestive of smooth muscle. The expression in these tissuesis strongest at E15.5 and E16.5 and decreases to almost backgroundlevels by P1.5. Adult expression is observed in a small number oftissues examined, including brain, submandibular gland, bladder, colon,small intestine, liver, spleen, and placenta. The signal in positivetissues is very weak in all but the mucosal epithelium of the bladderwhich has the strongest expression. There is no expression observed inthe following tested tissues: spinal cord, eye and harderian gland,white fat, brown fat, stomach, heart, kidney, adrenal gland, thymus,lymph node, lung, pancreas, skeletal muscle, and testes.

Uses of A259 Nucleic acids, Polypeptides, and Modulators Thereof

As human A259 was originally found in a LPS stimulated human primaryosteoblast library, A259 nucleic acids, proteins, and modulators thereofcan be used to modulate the proliferation, differentiation, and/orfunction of cells that form bone matrix, e.g., osteoblasts andosteoclasts, and can be used to modulate the formation of bone matrix.Thus A259 nucleic acids, proteins, and modulators thereof can be used totreat cartilage and bone associated diseases and disorders, and can playa role in bone growth, formation, and remodeling. Examples of cartilageand bone associated diseases and disorders include e.g., bone cancer,achondroplasia, myeloma, fibrous dysplasia, scoliosis, osteoarthritis,osteosarcoma, and osteoporosis.

As murine A259 was originally found in a bone marrow library, A259nucleic acids, proteins, and modulators thereof can be used to modulatethe proliferation, differentiation, and/or function of cells that appearin the bone marrow, e.g., stem cells (e.g., hematopoietic stem cells),and blood cells, e.g., erythrocytes, platelets, and leukocytes. ThusA259 nucleic acids, proteins, and modulators thereof can be used totreat bone marrow, blood, and hematopoietic associated diseases anddisorders, e.g., acute myeloid leukemia, hemophilia, leukemia, anemia(e.g., sickle cell anemia), and thalassemia.

As integrin family members play a role in immune response, A259 nucleicacids, proteins, and modulators thereof can be used to treat immunerelated disorders, e.g., immunodeficiency disorders (e.g., HIV), viraldisorders (e.g., infection by HSV), cell growth disorders, e.g., cancers(e.g., carcinoma, lymphoma, e.g., follicular lymphoma), autoimmunedisorders (e.g., arthritis, graft rejection (e.g., allograft rejection),T cell autoimmune disorders (e.g., AIDS)), and inflammatory disorders(e.g., bacterial or viral infection, psoriasis, septicemia, cerebralmalaria, inflammatory bowel disease, arthritis (e.g., rheumatoidarthritis, osteoarthritis), allergic inflammatory disorders (e.g.,asthma, psoriasis)).

As integrin family members play a role in cell growth, survival,proliferation, and migration, A259 nucleic acids, proteins, andmodulators thereof can be used to treat apoptotic disorders (e.g.,rheumatoid arthritis, systemic lupus erythematosus, insulin-dependentdiabetes mellitus) proliferative disorders (e.g., cancers, e.g., B cellcancers stimulated by TNF), and disorders abnormal vascularization(e.g., cancer). In addition, A259 nucleic acids, proteins, andmodulators thereof can also be used to promote vascularization(angiogenesis).

As integrins are cell adhesion molecules, A259 nucleic acids, proteins,and modulators thereof can be used to modulate disorders associated withadhesion and migration of cells, e.g., platelet aggregation disorders(e.g., Glanzmann's thromboasthemia, which is a bleeding disorderscharacterized by failure of platelet aggregation in response to cellstimuli), inflammatory disorders (e.g., leukocyte adhesion deficiency,which is a disorder associated with impaired migration of neutrophils tosites of extravascular inflammation), disorders associated with abnormaltissue migration during embryo development, and tumor metastasis.

A259 polypeptides, nucleic acids, and modulators thereof, can also beused to modulate the function, morphology, proliferation and/ordifferentiation of cells in the tissues in which it is expressed. Suchmolecules can be used to treat disorders associated with abnormal oraberrant metabolism or function of cells in the tissues in which it isexpressed. Tissues in which A259 is expressed, and disorders of whichA259 polypeptides, nucleic acids, and modulators thereof can be used totreat, include: bone (see disorders described herein); intestine (e.g.,ischemic bowel disease, infective enterocolitis, Crohn's disease); brain(e.g., cerebral edema, hydrocephalus, brain herniations, iatrogenicdisease (due to, e.g., infection, toxins, or drugs)); bladder (e.g.,cystitis (bladder infection), incontinence); liver (e.g., jaundice,hepatic failure, hepatic circulatory disorders (e.g., hepatic veinthrombosis and portal vein obstruction and thrombosis), hepatitis (e.g.,chronic active hepatitis, acute viral hepatitis, and toxic anddrug-induced hepatitis), cirrhosis (e.g., alcoholic cirrhosis, biliarycirrhosis, and hemochromatosis)); spleen (e.g., amyloidosis,Niemann-Pick disease, splenomegaly); placenta (e.g., toxemia ofpregnancy (e.g., preeclampsia and eclampsia, placentitis, spontaneousabortion); and neuronal tissue (e.g., epilepsy, muscular dystrophy, andneurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, and Huntington's disease).

A259 is expressed at a higher level in the liver of patients sufferingfrom liver fibrosis than in patients not suffering from liver fibrosis.Liver fibrosis, is caused by chronic injury to the liver arising from,e.g, alcohol abuse, drugs, viral infections (e.g., hepatitis B orhepatitis C), metabolic disorders (excessive iron or copper), autoimmuneattack on hepatocytes or the bile duct, and congenital disorders. Liverfibrosis is a reversible condition and injury can be present for monthsor years before significant scar tissue accumulates. However, liverfibrosis leads to cirrhosis, which is generally not reversible.

The liver has four major components: epithelial cells (hepatocytes),endothelial cells; tissue macrophages (Kupffer cells), and a stellatecells (a type of perivascular mesenchymal cell). In normal liver thespace between the epithelium and sinusoidal endothelium is filled with atype of extracellular matrix (ECM) that is somewhat similar to abasement membrane. In fibrosis the ECM undergoes a number of changes.The total content of collagens and other components increases severalfold. In addition, there is an increase in fibril-forming collagens.Stellate cells are the primary fibrogenic cell of the liver. In responseto injury, quiescent stellate cells are activated and becomeproliferative, fibrogenic, contractile myofibroblasts. Among the keymediators of these changes are: TGF-β1, MCP-1, PDGF, ET-1, and MMP-2.Kupffer cells, endothelial cells and hepatocytes produce factors whichstimulate stellate cell activation. As noted above, human A259 isexpressed at a high level in activated stellate cells. This result andthe increased expression of A259 in clinical liver fibrosis samples,suggest that A259 plays a role in liver fibrosis. Thus, A259 nucleicacids, polypeptides, and modulators thereof can be used to diagnose andtreat liver fibrosis as well as other types of fibrosis, e.g., kidneyfibrosis or lung fibrosis. Thus, liver fibrosis can be treated usingantibodies directed against A259, particularly the extracellular domainof A259, the I domain of A259, or a repeat domain of A259. Also usefulare polypeptides which include one or more repeat domains of A259 and/orthe I domain of A259.

Tables IV, V and VI below provide summaries of human A259 and murineA259 sequence information.

TABLE IV Summary of Sequence Information of Human A259 and Murine A259.ATCC Open Reading Accession Gene cDNA Frame Polypeptide No. Human A259SEQ ID SEQ ID NO: 146 SEQ ID 207190 NO: 145 NO: 147 Murine A259 SEQ IDSEQ ID NO: 164 SEQ ID 207191 NO: 163 NO: 165

TABLE V Summary of Domains of Human A259 and Murine A259. Signal MatureExtracellular Transmembrane Cytoplasmic Protein Sequence Protein DomainDomain Domain Human aa 1-22 aa 23-1188 aa 1-1141 aa 1142-1164 aa1165-1188 A259 SEQ ID SEQ ID SEQ ID SEQ ID NO: 159 SEQ ID NO: 160 NO:149 NO: 148 NO: 150 Murine aa 1-22 aa 23-1188 aa 1-1141 aa 1142-1164 aa1165-1188 A259 SEQ ID SEQ ID SEQ ID SEQ ID NO: 177 SEQ ID NO: 178 NO:167 NO: 166 NO: 168

TABLE VI Summary of Domains of Human A259 and Murine A259. Protein IDomain Repeat Domain Human aa 164-345 aa 39-74; 115-157; 367-392;421-455; 478-516; A259 SEQ ID 540-575; 602-640, SEQ ID NO: 152-158 NO:151 Murine aa 164-345 aa 39-74; 115-157; 367-392; 421-455; 478-516; A259SEQ ID 540-575; 602-640, SEQ ID NO: 170-176 NO: 169

Isolated Nucleic Acid Molecules of the Present Invention

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a polypeptide of the invention or a biologically activeportion thereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encoding apolypeptide of the invention and fragments of such nucleic acidmolecules suitable for use as PCR primers for the amplification ormutation of nucleic acid molecules.

As described below, one aspect of the invention pertains to isolatednucleic acids comprising nucleotide sequences encoding Delta3polypeptides, and/or equivalents of such polypeptides or nucleic acids.The “term equivalent” is understood to include nucleotide sequencesencoding functionally equivalent Delta3 polypeptides or functionallyequivalent peptides having an activity of a Delta3 protein such asdescribed herein. Equivalent nucleotide sequences also include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants, and will, therefore, include, forexample, sequences that differ from the nucleotide sequence of theDelta3 nucleic acid sequence shown in any of SEQ ID NOs: 1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45 due to the degeneracy of thegenetic code.

Another aspect of the invention pertains to isolated nucleic acidmolecules that encode FTHMA-070 or T85 proteins or biologically activeportions thereof, as well as nucleic acid molecules sufficient for useas hybridization probes to identify FTHMA-070 or T85-encoding nucleicacids (e.g., FTHMA-070 or T85 mRNA) and fragments for use as PCR primersfor the amplification or mutation of FTHMA-070 or T85 nucleic acidmolecules.

Another aspect of the invention pertains to isolated nucleic acidmolecules that encode Tango-77 proteins or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify Tango-77-encoding nucleic acids (e.g.,Tango-77 mRNA) and fragments for use as PCR primers for theamplification or mutation of Tango-77 nucleic acid molecules.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules that encode SPOIL proteins or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify SPOIL-encoding nucleic acids (e.g.,SPOIL mRNA) and fragments for use as PCR primers for the amplificationor mutation of SPOIL nucleic acid molecules.

Another aspect of the invention pertains to isolated nucleic acidmolecules that encode NEOKINE proteins or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify NEOKINE-encoding nucleic acids (e.g.,NEOKINE mRNA) and fragments for use as PCR primers for the amplificationor mutation of NEOKINE nucleic acid molecules.

Another aspect of the invention pertains to isolated nucleic acidmolecules that encode T129 proteins or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify T129-encoding nucleic acids (e.g., T129mRNA) and fragments for use as PCR primers for the amplification ormutation of T129 nucleic acid molecules.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences (preferably protein encoding sequences) which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatedFTHMA-070, T85, Tango 77, SPOIL, NEOKINE, T129 or A259 nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NOs: 1, 3, 24, 26, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 89,101, 104, 112, 115, 118, 121, 124, 137, 139, 145, 146, 163 or 164 or thenucleotide sequence of the cDNA of a clone deposited with the ATCC® asAccession Number 98348, 98807, 98883, 98984 or 98751 or a complementthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or a portion of thenucleic acid sequences of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 89, 101, 104, 112,115, 118, 121, 124, 137, 139, 145, 146, 163 or 164, or the nucleotidesequence of the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348, 98807, 98883, 98984 or 98751, as a hybridization probe,nucleic acid molecules of the invention can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook etal., eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

In another embodiment, a portion of the nucleic acid sequence of SEQ IDNO:89, for example, from nucleotides 1 to 15 or from nucleotides 447 or495 to 746, can used as a hybridization probe. In yet anotherembodiment, a portion of the nucleic acid sequence of SEQ ID NO:101, forexample, from nucleotides 1 to 280 or from nucleotides 390 to 1291, canbe used as a hybridization probe. In yet another embodiment, a portionof the nucleic acid sequence of SEQ ID NO:106, for example, fromnucleotides 1-371 or from 481-1377, can be used as a hybridizationprobe. In yet another embodiment, a portion of the nucleic acid sequenceof SEQ ID NO:112, for example, from nucleotides 225-364, from 96-575, orfrom 495-838, can be used as a hybridization probe.

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:89, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98883, or the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984, canbe isolated by the polymerase chain reaction (PCR) using syntheticoligonucleotide primers designed based upon the sequence of SEQ IDNO:89, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98883, or the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984.

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98751, can be isolated by the polymerase chainreaction (PCR) using synthetic oligonucleotide primers designed basedupon the sequence of SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ IDNO:124, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98751.

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 137, 139, 145, 146,163 or 164, or the nucleotide sequence of the cDNA of a clone depositedwith the ATCC® as Accession Number 98348, or 98807, or a portionthereof. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence under the conditions set forth herein, thereby forming a stableduplex.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:91. Thesequence of SEQ ID NO:91 corresponds to murine SPOIL-I cDNA. This cDNAcomprises sequences encoding the murine SPOIL-I protein (i.e., “thecoding region”, from nucleotides 135-428 of SEQ ID NO:89).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:92.The sequence of SEQ ID NO:92 corresponds to murine SPOIL-I cDNA. ThiscDNA comprises sequences encoding the mature SPOIL-I protein (i.e., fromnucleotides 186-428 of SEQ ID NO:89 after the signal sequence has beencleaved).

In yet another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:89. Thesequence of SEQ ID NO:89 corresponds coding and noncoding regions ofmurine SPOIL-I cDNA. This cDNA comprises sequences encoding the murineSPOIL-I protein (i.e., “the coding region”, from nucleotides 135-428)and noncoding regions (i.e., from nucleotides 1-134 and from nucleotides429-746).

In yet another embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:112. Thesequence of SEQ ID NO:112 corresponds coding and noncoding regions ofmurine SPOIL-II cDNA. This cDNA comprises sequences encoding the murineSPOIL-II protein (i.e., “the coding region”, from nucleotides 96-575)and noncoding regions (i.e., from nucleotides 1-95 and from nucleotides576-838).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:101.The sequence of SEQ ID NO:101 corresponds to the human SPOIL-I cDNA.This cDNA comprises sequences encoding the human SPOIL-I protein (i.e.,“the coding region”, from nucleotides 124 to 630), as well as 5′untranslated sequences (nucleotides 1 to 123) and 3′ untranslatedsequences (nucleotides 631 to 1291). Alternatively, the nucleic acidmolecule can comprise only the coding region of SEQ ID NO:101 (e.g.,nucleotides 124 to 630).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:104.The sequence of SEQ ID NO:104 corresponds to the human SPOIL-II cDNA.This cDNA comprises sequences encoding the human SPOIL-II protein (i.e.,“the coding region”, from nucleotides 98-721, as well as 5′ untranslatedsequences (nucleotides 1-97 and 3′ untranslated sequences (nucleotides722-1377). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:104 (e.g., nucleotides 98-721).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:89, SEQ ID NO:101, SEQ IDNO:104, SEQ ID NO:112, the DNA insert of the plasmid deposited with ATCCas Accession Number 98883, or the DNA insert of the plasmid depositedwith ATCC as Accession Number 98984, or a portion of any of thesenucleotide sequences. A nucleic acid molecule which is complementary tothe nucleotide sequence shown in SEQ ID NO:89, SEQ ID NO:101, SEQ IDNO:104, SEQ ID NO:112, the DNA insert of the plasmid deposited with ATCCas Accession Number 98883, or the DNA insert of the plasmid depositedwith ATCC as Accession Number 98984, is one which is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO:89, SEQ IDNO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the DNA insert of theplasmid deposited with ATCC as Accession Number 98984, such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:89, SEQ IDNO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the DNA insert of theplasmid deposited with ATCC as Accession Number 98984, or a complementthereof, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50-55%, 60-65%, preferably at least about 70-75%, morepreferable at least about 80-85%, and even more preferably at leastabout 90-95% or more identical to the nucleotide sequences shown in SEQID NO:89, the nucleotide sequence shown in SEQ ID NO:101, the nucleotidesequence shown in SEQ ID NO:104, the nucleotide sequence shown in SEQ IDNO:112, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, the DNA insert of theplasmid deposited with ATCC as Accession Number 98984, or a portion ofany of these nucleotide sequences.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:115. Thesequence of SEQ ID NO:115 corresponds to the human NEOKINE-1 cDNA. ThiscDNA comprises sequences encoding the human NEOKINE-1 protein (i.e.,“the coding region”, from nucleotides 97-393), as well as 5′untranslated sequences (nucleotides 1-97) and 3′ untranslated sequences(nucleotides 394-1564). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:115 (e.g., nucleotides97-393, corresponding to SEQ ID NO:117).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:118.The sequence of SEQ ID NO:118 corresponds to a murine NEOKINE-1 cDNA.This cDNA comprises sequences encoding the murine NEOKINE-1 protein(i.e., “the coding region”, from nucleotides 212-508), as well as 5′untranslated sequences (nucleotides 1-211) and 3′ untranslated sequences(nucleotides 509-1656). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:118 (e.g., nucleotides212-508, corresponding to SEQ ID NO:120).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:121.The sequence of SEQ ID NO:121 corresponds to a rat NEOKINE-1 cDNA. ThiscDNA comprises sequences encoding at least 79 amino acid residues of therat NEOKINE-1 protein (i.e., “the coding region”, from nucleotides1-234), as well as 3′ untranslated sequences (nucleotides 235-1372).Alternatively, the nucleic acid molecule can comprise only the codingregion of SEQ ID NO:121 (e.g., nucleotides 235-1372, corresponding toSEQ ID NO:123).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:124.The sequence of SEQ ID NO:124 corresponds to a macaque NEOKINE-1 cDNA.This cDNA comprises sequences encoding at least 94 amino acid residuesof the macaque NEOKINE-1 protein (i.e., “the coding region”, fromnucleotides 3-284), as well as 3′ untranslated sequences (nucleotides285-1458). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:124 (e.g., nucleotides 285-1458,corresponding to SEQ ID NO:136).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:115, SEQ ID NO:118, SEQ IDNO:121, SEQ ID NO:124, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98751, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:115, SEQ IDNO:118, SEQ ID NO:121, SEQ ID NO:124, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98751,is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98751, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:115, SEQ ID NO:118, SEQ IDNO:121, SEQ ID NO:124, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98751, therebyforming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 60-65%, preferably at least about 70-75%, more preferable atleast about 80-85%, and even more preferably at least about 90-95%,96-97%, 98-99% or more homologous to the nucleotide sequences shown inSEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98751, or a portion of any of these nucleotidesequences.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding a full-length polypeptide ofthe invention for example, a fragment which can be used as a probe orprimer or a fragment encoding a biologically active portion of apolypeptide of the invention. The nucleotide sequence determined fromthe cloning one gene allows for the generation of probes and primersdesigned for use in identifying and/or cloning homologs in other celltypes, e.g., from other tissues, as well as homologs from other mammals.The probe/primer typically comprises substantially purifiedoligonucleotide.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350 or 400 consecutive nucleotides of the sense oranti-sense sequence of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37,39, 41, 43 or 45, or the nucleotide sequence of the cDNA of a clonedeposited with the ATCC® as Accession Number 98348, or of anaturally-occurring mutant of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33,35, 37, 39, 41, 43 or 45, or the nucleotide sequence of the cDNA of aclone deposited with the ATCC® as Accession Number 98348.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350 or 400 consecutive nucleotides of the sense oranti-sense sequence of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:58, or of a naturally occurring mutant of SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:57, SEQ ID NO:58.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350 or 400 consecutive nucleotides of the sense oranti-sense sequence of SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ IDNO:80, or the cDNA of ATCC 98807. Alternatively, the oligonucleotide cantypically comprise a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably about 25, morepreferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400consecutive nucleotides of the sense or anti-sense sequence of anaturally occurring mutant of SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76,SEQ ID NO:80, or the cDNA of ATCC 98807.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 40, 50 or 75 consecutivenucleotides of a sense sequence of SEQ ID NO:89, SEQ ID NO:101, SEQ IDNO:104, SEQ ID NO:112, the DNA insert of the plasmid deposited with ATCCas Accession Number 98883, or the DNA insert of the plasmid depositedwith ATCC as Accession Number 98984, of an anti-sense sequence of SEQ IDNO:89, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98883, or the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984, orof a naturally occurring mutant of either SEQ ID NO:89, SEQ ID NO:101,SEQ ID NO:104, SEQ ID NO:112, the DNA insert of the plasmid depositedwith ATCC as Accession Number 98883, or the DNA insert of the plasmiddeposited with ATCC as Accession Number 98984.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 40, 50 or 75 consecutivenucleotides of a sense sequence of SEQ ID NO:115, SEQ ID NO:118, SEQ IDNO:121, SEQ ID NO:124, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98751, of ananti-sense sequence of SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQID NO:124, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98751, or of a naturallyoccurring mutant of SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ IDNO:124, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98751. In an exemplaryembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which is greater that 500 nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124,or the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC as Accession Number 98751.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350 or 400 consecutive nucleotides of the sense oranti-sense sequence of SEQ ID NO:137 or SEQ ID NO:139, or of a naturallyoccurring mutant of SEQ ID NO:137 or SEQ ID NO:139.

The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350 or 400 consecutive nucleotides of the sense oranti-sense sequence of SEQ ID NO:145, 146, 163, or 164 or of a naturallyoccurring mutant of SEQ ID NO: 145, 146, 163, or 164.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences encoding the sameprotein molecule encoded by a selected nucleic acid molecule. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit for identifying cells ortissues which mis-express the protein, such as by measuring levels of anucleic acid molecule encoding the protein in a sample of cells from asubject, e.g., detecting mRNA levels or determining whether a geneencoding the protein has been mutated or deleted.

A nucleic acid fragment encoding a biologically active portion of apolypeptide of the invention can be prepared by isolating a portion ofany of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, orthe amino acid sequence encoded by the cDNA of a clone deposited withthe ATCC® as Accession Number 98348 expressing the encoded portion ofthe polypeptide protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the polypeptide.

A nucleic acid fragment encoding a “biologically active portion ofFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259” can beprepared by isolating a portion of SEQ ID NO:53, 55, 57, 58, 71, 73, 76,80, 89, 101, 104, 112, 115, 118, 121, 124, 137, 139, 146 or 164, or thenucleotide sequence of the cDNA of ATCC 98807, 98883, 98984 or 98751,which encodes a polypeptide having a FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 biological activity, expressing the encodedportion of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 89, 101,104, 112, 115, 118, 121, 124, 137, 139, 145, 146, 163 or 164, or thenucleotide sequence of the cDNA of a clone deposited with the ATCC® asAccession Number 98348, 98807, 98883, 98984 or 98751, due to degeneracyof the genetic code and thus encode the same protein as that encoded bythe nucleotide sequence of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 89, 101, 104, 112,115, 118, 121, 124, 137, 139, 145, 146, 163 or 164, or the nucleotidesequence of the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348, 98807, 98883, 98984 or 98751.

In addition to the nucleotide sequences of SEQ ID NOs: 1, 3, 24, 26, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 89,101, 104, 112, 115, 118, 121, 124, 137, 139, 145, 146, 163 or 164, orthe nucleotide sequence of the cDNA of a clone deposited with the ATCC®as Accession Number 98348, 98807, 98883, 98984 or 98751, it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequence may exist within apopulation (e.g., the human population). Such genetic polymorphisms mayexist among individuals within a population due to natural allelicvariation.

Such allelic variants of a polymorphic region of a Delta3 gene are alsoincluded as part of the present invention. For example, the human genefor Delta3 was mapped to chromosome 15, between markers D15S1244 andD15S144, and therefore, human Delta3 family members can includenucleotide sequence polymorphisms (e.g., nucleotide sequences that varyfrom SEQ ID NO: 1,3, 24, 26, 27, 29, 31, 33, 35, or 37) that map to thischromosome 15 region (i.e., between framework regions D15S1244 andD15S144), as well as polypeptides encoded therefrom.

An allele is one of a group of genes which occur alternatively at agiven genetic locus. As used herein, the terms “gene” and “recombinantgene” refer to nucleic acid molecules comprising an open reading frameencoding a Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 protein, preferably a mammalian Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 protein. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention sequence that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of theprotein. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence without altering the biological activity, whereasan “essential” amino acid residue is required for biological activity.For example, amino acid residues that are not conserved or onlysemi-conserved among homologs of various species may be non-essentialfor activity and thus would be likely targets for alteration.Alternatively, amino acid residues that are conserved among theorthologs of various species (e.g., murine and human) may be essentialfor activity and thus would not be likely targets for alteration.

In one embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 786 is an cytosine (C) (SEQ ID NO: 1). In thisembodiment, the amino acid at position 150 is a alanine (A) (SEQ ID NO:2). In an alternative embodiment, a species variant of human Delta3 hasa nucleotide at position 786 which is a thymidine (T) (SEQ ID NO: 33).In this embodiment, the amino acid at position 150 is valine (V) (SEQ IDNO: 34), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 594 is a cytosine (C) (SEQ ID NO: 1). In thisembodiment, the amino acid at position 86 is threonine (T) (SEQ ID NO:2). In an alternative embodiment, a species variant of human Delta3 hasa nucleotide at position 594 which is a guanine (G) (SEQ ID NO: 35). Inthis embodiment, the amino acid at position 86 is serine (S) (SEQ ID NO:36), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of human Delta3, wherein thenucleotide at position 883 is a thymidine (T) (SEQ ID NO: 1). In thisembodiment, the amino acid at position 182 is aspartate (D) (SEQ ID NO:2). In an alternative embodiment, a species variant of human Delta3 hasa nucleotide at position 883 which is an adenine (A) (SEQ ID NO: 37). Inthis embodiment, the amino acid at position 182 is glutamate (E) (SEQ IDNO: 38), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, thenucleotide at position 49 is cytosine (C) (SEQ ID NO: 24). In thisembodiment, the amino acid at position 4 is alanine (A) (SEQ ID NO: 25).In an alternative embodiment, a species variant of mouse Delta3 has anucleotide at position 49 which is thymidine (T) (SEQ ID NO: 39). Inthis embodiment, the amino acid at position 4 is valine (V) (SEQ ID NO:40), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, thenucleotide at position 51 is thymidine (T) (SEQ ID NO: 24). In thisembodiment, the amino acid at position 5 is serine (S) (SEQ ID NO: 25).In an alternative embodiment, a species variant of mouse Delta3 has anucleotide at position 51 which is a adenine (A) (SEQ ID NO: 41). Inthis embodiment, the amino acid at position 5 is threonine (T) (SEQ IDNO: 42), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, thenucleotide at position 109 is guanine (G) (SEQ ID NO: 24). In thisembodiment, the amino acid at position 24 is arginine (R) (SEQ ID NO:25). In an alternative embodiment, a species variant of mouse Delta3 hasa nucleotide at position 109 which is adenine (A) (SEQ ID NO: 43). Inthis embodiment, the amino acid at position 24 is histidine (H) (SEQ IDNO: 44), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, wherein thenucleotide at position 130 is a thymidine (T) (SEQ ID NO: 24). In thisembodiment, the amino acid at position 31 is phenylalanine (F) (SEQ IDNO: 25). In an alternative embodiment, a species variant of mouse Delta3has a nucleotide at position 130 which is adenine (A) (SEQ ID NO: 45).In this embodiment, the amino acid at position 31 is tyrosine (Y) (SEQID NO: 46), i.e., a conservative substitution.

The invention also pertains to nucleic acid molecules encoding apolypeptide of the invention that contain changes in amino acid residuesthat are not essential for activity. Such polypeptides differ in aminoacid sequence from SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348 yet retain biologicalactivity. In one embodiment, the isolated nucleic acid molecule includesa nucleotide sequence encoding a protein that includes an amino acidsequence that is at least 65%, 75%, 85%, 95%, or 98% identical to theamino acid sequence of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348.

Moreover, nucleic acid molecules encoding proteins of the invention fromother species (homologs), which have a nucleotide sequence which differsfrom that of the human protein described herein are intended to bewithin the scope of the invention. Nucleic acid molecules correspondingto natural allelic variants and homologs of a cDNA of the invention canbe isolated based on their identity to the human nucleic acid moleculedisclosed herein using the human cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. For example, a cDNA encoding asoluble form of a membrane-bound protein of the invention isolated basedon its hybridization to a nucleic acid molecule encoding all or part ofthe membrane-bound form. Likewise, a cDNA encoding a membrane-bound formcan be isolated based on its hybridization to a nucleic acid moleculeencoding all or part of the soluble form.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 53, 55, 57, 58, 71, 73, 76, 80, 89, 101, 104, 112, 115, 118,121, 124, 137, 139, 145, 146, 163 or 164, or the cDNA of ATCC® asAccession Number 98348, 98807, 98883, 98984 or 98751 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 480 (500, 550, 600, 650, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or 2050)nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising the nucleotide sequence, preferably thecoding sequence, of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37,39, 41, 43 or 45, or the nucleotide sequence of the cDNA of a clonedeposited with the ATCC® as Accession Number 98348, or complementthereof.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence, ofSEQ ID NO:53, 55, 57 or 58.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, or 989) nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence, preferably the coding sequence, of SEQ ID NO:71,73, 76, 80, or the cDNA of ATCC 98807.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:89, 101, 104, 112 or the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98883 or 98984, or acomplement thereof. In other embodiments, the nucleic acid is at least30, 50, 100, 250, 300, 400, 500, 600 or 700 nucleotides in length.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:115, 118, 121, 124, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98751. In other embodiment, the nucleic acid is atleast 30, 50, 100, 250 or 500 nucleotides in length.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence, ofSEQ ID NO:137 or 139

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 400 (450, 500, 550, 600, 650, 700, 800, 900,1000, 2000, or 3000) nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence, preferably the coding sequence, of SEQ ID NO:145 or163, or a complement thereof.

Preferably, such nucleic acid molecules, “specifically hybridize” or“specifically detect” a nucleic acid molecule of the invention byexhibiting the ability to hybridize to at least approximately 6, 12, 20,30, 50, 100, 150, 200, 300, 350, 400 or 425 consecutive nucleotides of aDelta3 nucleotide sequence designated in one of SEQ ID Nos:1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotide sequence ofthe cDNA of a clone deposited with the ATCC® as Accession Number 98348,or a sequence complementary thereto, such that more than 5, 10 or 20times more hybridization (utilizing hybridization conditions describedabove), preferably more than 50 times more hybridization, and even morepreferably more than 100 times more hybridization than occurs relativeto hybridization to a cellular nucleic acid (e.g., mRNA or genomic DNA)encoding a protein other than a Delta3 protein as defined herein.

Delta3 nucleic acids can encode polypeptides that are at least 55%identical to an amino acid sequence of SEQ ID NOs: 2, 25, 28, 30, 32,34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded by thecDNA of a clone deposited with the ATCC® as Accession Number 98348.Nucleic acids which encode polypeptides which are at least about 72%,and even more preferably at least about 80%, 85%, 90%, 95%, or 98%similar with an amino acid sequence represented in SEQ ID NO: 2, 25, 28,30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encodedby the cDNA of a clone deposited with the ATCC® as Accession Number98348 are also within the scope of the invention.

In one embodiment, the nucleic acid of the present invention encodes apolypeptide having an overall amino acid sequence similarity of at leastabout 72%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99% with theamino acid sequence shown in SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38,40, 42, 44 or 46. In a preferred embodiment, the nucleic acid encodes aprotein comprising the amino acid set forth in SEQ ID NOs: 2, 25, 28,30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encodedby the cDNA of a clone deposited with the ATCC® as Accession Number98348. Preferably, the nucleic acid includes all or a portion of thenucleotide sequence corresponding to the coding region of SEQ ID Nos: 1,3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the nucleotidesequence of the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348.

The nucleic acids of the invention can encode a Delta3 protein from anyspecies, including insects. Preferred nucleic acids encode vertebrateDelta3 proteins. Even more preferred nucleic acids encode mammalianDelta3 proteins including primate Delta3 proteins, e.g., human Delta3proteins, and murine Delta3 proteins. Other nucleic acids of theinvention can encode avian, equine, canine, feline, bovine or porcineDelta3 proteins.

In a preferred embodiment of the invention, the nucleic acid encodes apolypeptide comprising an extracellular domain of Delta3, e.g., human ormouse Delta3 including allelic variants having SEQ ID NOs: 2, 25, 28,30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encodedby the cDNA of a clone deposited with the ATCC® as Accession Number98348. Accordingly, preferred nucleic acids encode a polypeptidecomprising about amino acid 1 to about amino acid 529 of SEQ ID NO: 2,28, 30, 32, 34, 36 or 38, or alternatively about amino acid 1 to aboutamino acid 530 of SEQ ID NO: 25, 40, 42, 44, or 46.

Other preferred nucleic acids encode a polypeptide corresponding to anextracellular domain of Delta3 lacking the signal peptide, e.g., apolypeptide comprising about amino acid 18 to about amino acid 529 ofSEQ ID NO: 2 or about amino acid 18 to about amino acid 530 of SEQ IDNO: 25. Yet other preferred nucleic acids encode a polypeptidecomprising at least one of the conserved motifs in the extracellulardomain of Delta3, e.g., a DSL motif (for example, amino acids 173-217 ofSEQ ID NO: 2 or amino acids 174-218 of SEQ ID NO: 25) or an EGF-likemotif (for example, EGF-like 1, amino acids 222-250 of SEQ ID NO: 2), oralso for example amino acids 223-251 of SEQ ID NO: 25. AdditionalEGF-like domains are from amino acids 253-281, 288-321, 328-359,366-399, 411-437, 444-475, and 484-517 of SEQ ID NO: 2 and 254-282,289-322, 329-360, 367-400, 412-438, 445-476, and 485-518 of SEQ ID NO:25.

In one embodiment, the nucleic acid encodes a protein having at leastone EGF-like motif. In other embodiments, the nucleic acid encodesproteins having at least 2, at least 3, at least 4, at least 5, at least6, at least 7, or 8 EGF-like domains or amino acids 223-251, 254-282,289-322, 329-360, 367-400, 412-438, 445-476, and 485-518 of SEQ ID NO:25.

The polypeptide encoded by a nucleic acid encoding any of these numbersof EGF-like domains can further comprise an amino acid sequence encodinga DSL motif.

The DSL region or motif is shared by all known members of the family ofpresumed ligands of Notch-like proteins (Delta1 and Serrate inDrosophila; Lag-2 and Apx-1 in Caenorhabditis elegans) (Henderson et al.(1994) Development 120:2913; Tax et al. (1994) Nature 368:150; Fleminget al. (1990) Genes Dev. 4:2188; Thomas et al. (1991) Development11:749; Mello et al. (1994) Cell 77:95). The DSL motif is located in theamino terminal portion of the protein, i.e., extracellular, which isclosely related to a similar domain in the Drosophila Delta1 protein andwhich has been described as being necessary and sufficient for in vitrobinding to Notch (Henrique et al. (1995) Nature 375:787; Muskavitch(1994) Dev. Biol. 166:415).

In one embodiment, a nucleic acid of the invention encodes a polypeptidethat comprises a human or mouse Delta3 DSL domain and which is capableof binding a receptor. A Delta3 DSL domain conforms to the following DSLconsensus sequence:X-X-C-X-X-X-Y-[FY]-G-X-X-C-X-X-X-C-[HR]-X-R-X-D-X-F-G-[RH]-X-X-C-X-X-X-G-X-X-X-C-X-X-X-C-X-X-G-W-X-G-X-Y-C(SEQ ID NO:23), wherein all amino acids are indicated according to theiruniversal single letter designation, brackets indicate that the aminoacid at that position is selected from one of the amino acids within thebrackets, and “X” designates any amino acid, and wherein the DSL domainis at least 60%, or more preferably at least 65%, 70%, 75%, 80%, 85%, ormost preferably 90%, 95%, 98% or 100% identical, i.e., no gaps in thesequence, to the human Delta3 polypeptide sequence from amino acids173-217 of SEQ ID NO:2. In another embodiment, a Delta3 DSL domainconforms to the above-described DSL consensus sequence and is at least60%, or more preferably at least 65%, 70%, 75%, 80%, 85%, or mostpreferably 90%, 95%, 98% or 100% identical to the mouse Delta3 DSLsequence from amino acids 174-218 of SEQ ID NO: 25.

In another embodiment, a nucleic acid of the invention encodes Delta3DSL domain has a cysteine at amino acid positions 176, 185, 189, 201,209, and 217 of SEQ ID NO: 2, and is at least 60%, or more preferably atleast 65%, 70%, 75%, 80%, 85%, or most preferably 90%, 95%, 98% or 100%identical to the human Delta3 polypeptide sequence from amino acids173-217 of SEQ ID NO:2. In another embodiment, a Delta3 DSL domain has acysteine at amino acid positions 177, 186, 190, 202, 210, and 218 of SEQID NO: 25, and is at least 60%, or more preferably at least 65%, 70%,75%, 80%, 85%, or most preferably 90%, 95%, 98% or 100% identical to themouse Delta3 DSL sequence from amino acids 174-218 of SEQ ID NO: 25.

In one embodiment, a nucleic acid of the invention encodes a polypeptidethat comprises a human or mouse Delta3 EGF-like domain. An EGF-likedomain has the following consensus sequence:C-X₄₋₈-C-X₁₋₂-G-X-C-X₅₋₉-[WFY]-X-C-X-C-X₂₋₄-G-[WFY]-G-X₁₋₃-[FY]-C (SEQID NO:52), wherein all amino acids are indicated according to theiruniversal single letter designation, brackets indicate that the aminoacid at that position is selected from one of the amino acids within thebrackets, and “X” designates any amino acid. The numbers in subscriptnext to an amino acid position indicate a range of possible amino acids,for example, C-X₅₋₉-C indicates that there is a cysteine followed by any5 to 9 amino acids followed by a cysteine. In one embodiment, anEGF-like domain of the invention is at least 75%, or more preferably atleast 80%, 85%, or most preferably 90%, 95%, 98% or 100% identical,i.e., no gaps in the sequence, to the human Delta3 polypeptide sequencefrom amino acids 222-250, amino acids 253-281, amino acids 288-321,amino acids 328-359, amino acids 366-399, amino acids 411-437, aminoacids 444-475, and amino acids 484-517 of SEQ ID NO: 2. In anotherpreferred embodiment, a Delta3 EGF-like domain conforms to theabove-described EGF-like consensus sequence and is at least 60%, or morepreferably at least 75%, 80%, 85%, or most preferably 90%, 95%, 98% or100% identical to the mouse Delta3 DSL sequence from amino acids223-251, amino acids 254-282, amino acids 289-322, amino acids 329-360,amino acids 367-400, amino acids 412-438, amino acids 445-476, and aminoacids 485-518 of SEQ ID NO: 25.

In another embodiment, a nucleic acid of the invention encodes Delta3EGF-like domain is at least 75%, or more preferably at least 80%, 85%,or most preferably 90%, 95%, 98% or 100% identical to the human Delta3polypeptide sequence from amino acids 222-250, amino acids 253-281,amino acids 288-321, amino acids 328-359, amino acids 366-399, aminoacids 411-437, amino acids 444-475, or amino acids 484-517 of SEQ ID NO:2. In another embodiment, a Delta3 EGF-like domain is at least 75%, ormore preferably at least 80%, 85%, or most preferably 90%, 95%, 98% or100% identical to the mouse Delta3 EGF-like domain sequence from aminoacids 223-251, amino acids 254-282, amino acids 289-322, amino acids329-360, amino acids 367-400, amino acids 412-438, amino acids 445-476,or amino acids 485-518 of SEQ ID NO: 25.

Polypeptides encoded by any of the above-described nucleic acids can besoluble. Preferred soluble peptides comprise at least a portion of theextracellular domain of a Delta3 protein. Even more preferred solublepolypeptides comprise an amino acid sequence corresponding to aboutamino acid 1 to about amino acid 529 of SEQ ID NO: 2, corresponding toabout amino acid 18 to about amino acid 529 of SEQ ID NO: 2 or a homologthereof. Alternatively, an extracellular domain is comprised of aboutamino acid 1 to about amino acid 530 of SEQ ID NO: 25, about amino acid18 to about amino acid 530 of SEQ ID NO: 25 or a homolog thereof.

Yet other preferred soluble Delta3 polypeptides comprise at least oneEGF-like domain. Such polypeptides may in addition comprise a DSL domainand optionally a signal peptide.

In another embodiment, nucleic acids encode a Delta3 polypeptide as partof a fusion protein. A preferred fusion protein is a Delta3Immunoglobulin (Ig) fusion protein, or alternatively, a Delta3 portionas described above fused to Ig. Such fusion proteins can comprise atleast a portion of the extracellular domain of a Delta3 domain. Aportion can be any portion of at least about 10 amino acids, such as theportions described above. Nucleic acids encoding such fusion proteinscan be prepared, e.g., as described in U.S. Pat. No. 5,434,131.

Alternatively, polypeptides encoded by the nucleic acid of the inventioncan be membrane bound. Membrane bound polypeptides of the inventionpreferably comprise a transmembrane domain. As used herein, a“transmembrane domain” refers to an amino acid sequence having at leastabout 20 to 25 amino acid residues in length and which contains at leastabout 65-70% hydrophobic amino acid residues such as alanine, leucine,isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. Thetransmembrane domain can be from a Delta3 protein, such as atransmembrane domain comprising amino acid 530 to amino acid 553 of SEQID NO: 2, or amino acid 531 to amino acid 554 of SEQ ID NO: 25.

In a one embodiment, a nucleic acid of the invention encodestransmembrane domain contains at least about 15 to 30 amino acidresidues, preferably about 20-25 amino acid residues, and has at leastabout 60-80%, more preferably 65-75%, and more preferably at least about70% hydrophobic residues from about amino acid 530 to about amino acid553 of SEQ ID NO: 2, or from about amino acid 531 to about amino acid554 of SEQ ID NO: 25.

Alternatively, the transmembrane domain can be from another membraneprotein, such as to produce a chimeric membranous Delta3 protein. Yetother polypeptides of the invention can be intracellular proteins.Accordingly, also within the scope of the invention are proteins whichdo not comprise a transmembrane domain. Other proteins of the inventiondo not include an extracellular domain. Additional proteins of theinvention do not include an extracellular domain nor a transmembranedomain.

Polypeptides encoded by the nucleic acid of the invention can comprise acytoplasmic domain. In a preferred embodiment, a nucleic acid of theinvention encodes a polypeptide comprising a Delta3 cytoplasmic domain.In an even more preferred embodiment, the cytoplasmic domain has anamino acid sequence corresponding to a sequence from about amino acid554 to about amino acid 685 of SEQ ID NO: 2, or a portion thereof, orfrom about amino acid 555 to about amino acid 686 of SEQ ID NO: 25.

In yet other preferred embodiments, the nucleic acid of the inventionencodes a polypeptide comprising at least one domain of a Delta3 proteinselected from the group consisting of: a signal peptide, a DSL motif, anEGF-like domain, a transmembrane domain, and a cytoplasmic domain. Thepolypeptide of the invention can comprise several of these domains froma Delta3 protein.

Alternatively, a nucleic acid of the invention encodes a polypeptidethat can be a chimeric protein, i.e., comprised of at least oneconserved domain of SEQ ID NO: 2, 25, 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46, or the amino acid sequence encoded by the cDNA of a clonedeposited with the ATCC® as Accession Number 98348 and at least oneconserved domain from a polypeptide other than a Delta3 protein.Accordingly, in one embodiment, a nucleic acid of the invention encodesa Delta3 polypeptide of 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46,or the amino acid sequence encoded by the cDNA of a clone deposited withthe ATCC® as Accession Number 98348 wherein, for example, the DSL motiffrom amino acids 173-217 of SEQ ID NO: 2, are replaced with amino acidsfrom a comparable DSL domain of a Delta-like protein other than Delta3.

In yet another embodiment, the nucleic acid encodes a Delta3 proteinhaving a signal peptide from a protein other than a Delta3 protein. Alsowithin the scope of the invention are Delta3 nucleic acids which encodea Delta3 polypeptide, wherein the cytoplasmic domain is other than aDelta3 cytoplasmic domain. In addition, the invention contemplates aDelta3 nucleic acid molecule, wherein the nucleic acid encodes a Delta3polypeptide with a cytoplasmic domain and a extracellular domain from aprotein other than Delta3.

Delta-like proteins other than Delta3 proteins can be, e.g., toporythmicproteins. “Toporythmic proteins” is intended to include Notch, Delta,Serrate, Enhancer of Split, Deltex, and other members of this family ofproteins sharing structural similarities. (See e.g., InternationalPatent Publication Nos. WO 97/01571 (Jan. 16, 1997); WO 92/19734 (Nov.12, 1992) and WO 94/07474 (Apr. 14, 1994)).

Nucleic acids encoding polypeptides having an amino acid sequence thatis homologous to any of the above described portions of SEQ ID NO: 2,25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acid sequenceencoded by the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348 are also within the scope of the invention. Preferrednucleic acids of the invention encode polypeptides comprising an aminoacid sequence which are at least about 70%, at least about 75%, at leastabout 80%, or at least about 85% identical to the amino acid sequence ofany of the Delta3 domains. Even more preferred nucleic acids of theinvention encode polypeptides comprising an amino acid sequence whichare at least about 90%, at least about 95%, at least about 98%, or atleast about 99% identical to the amino acid sequence of any of theDelta3 domains.

In one embodiment, the nucleic acid, e.g., cDNA, encodes a peptidehaving at least one activity of the subject Delta3 polypeptide, such asthe ability to bind to a Delta3 interacting molecule, such as a Delta3receptor e.g., Notch. Non-limiting examples of binding assays for Delta3interaction with a Delta3 interacting molecule include: measuringinteraction of Delta3 polypeptides of the invention with a Delta3interacting molecule, such as for example Notch, include binding assaysinvolving soluble forms of Delta3 and a Delta3 interacting molecule,measuring a Delta3 domain, e.g., the DSL domain, binding to a Delta3interacting molecule, measuring Delta3 binding to receptors expressed oncells, and measuring soluble Delta3 binding to an immobilized Delta3interacting molecule, i.e., solid-phase binding assays. Specificexamples of these assays are set forth in Shimizu et al. (1999) J. Biol.Chem. 274:32961-32969.

Additional molecules, e.g., polypeptides or peptides, capable ofinteracting with a Delta3 protein or fragment thereof can be identifiedby various methods, e.g., methods based on binding assays. For example,various types of expression libraries can be screened with a Delta3protein or portion thereof. A two-hybrid system can be used to isolatecytoplasmic proteins interacting with the cytoplasmic domain of Delta3.Portions of Delta3 proteins which are capable of interacting with aligand can be determined by preparing fragments of Delta3 proteins andscreening these fragments for those that are capable of interacting withthe ligand.

Based at least in part on the observation that the N-terminal portion ofDrosophila Delta protein, which contains a DSL domain and EGF-likedomain, is necessary and sufficient for in vitro binding to Notch(Henrique et al. (1995) Nature 375:787; Muskavitch (1994) Dev. Biol.166:415), it is likely that the domain of Delta3 proteins capable ofinteracting with a ligand includes the DSL domain and/or at least aportion of the EGF-like domain. However, other portions of theextracellular domain of Delta3 could be necessary for binding to atleast some Delta3 ligands.

In other preferred embodiments, the subject Delta3 polypeptide canmodulate proliferation and/or differentiation or cell death of specifictarget cells, e.g., neural cells or endothelial cells. Assays fordetermining that a Delta3 polypeptide has at least one activity of aDelta3 protein are described infra.

Still other preferred nucleic acids of the present invention encode aDelta3 polypeptide which includes a polypeptide sequence correspondingto all or a portion of amino acid residues in SEQ ID NO: 2, 25, 28, 30,32, 34, 36, 38, 40, 42, 44 or 46, e.g., at least 2, 5, 10, 25, 50, 100,150 or 200 amino acid residues of that region. Preferred nucleic acidsencode a polypeptide comprising at least two consecutive amino acidresidues from about amino acid 1 to about amino acid 570 of the aminoacid sequence set forth in SEQ ID NO: 2 or from about amino acid 1 toabout amino acid 571 of the amino acid sequence set forth in SEQ ID NO:25.

Yet other preferred nucleic acids encode a polypeptide comprising atleast about 3, at least about 5, at least about 10, at least about 15,at least about 20, or at least about 25 consecutive amino acids fromabout amino acid 1 to about amino acid 575 set forth in SEQ ID NO: 2,from about amino acid 18 to about amino acid 575 set forth in SEQ ID NO:2, from about amino acid 1 to about amino acid 576 set forth in SEQ IDNO: 25, or from about amino acid 18 to about amino acid 576 set forth inSEQ ID NO: 25.

The invention further provides for nucleic acids encoding a polypeptidehaving an amino acid sequence which is at least about 70%, preferably atleast about 80%, and most preferably at least about 90% to at leastabout 10 consecutive amino acids set forth in SEQ ID NOs: 2, 25, 28, 30,32, 34, 36, 38, 40, 42, 44 or 46, or at least about 10 consecutive aminoacids from a portion of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46. In one embodiment, the portion corresponds to about aminoacid 1 to about amino acid 575 of SEQ ID NO: 2, about amino acid 18 toabout amino acid 575 of SEQ ID NO: 2, from about amino acid 1 to aboutamino acid 576 set forth in SEQ ID NO: 25, or from about amino acid 1 toabout amino acid 576 set forth in SEQ ID NO: 25. Coding nucleic acidmolecules of the invention preferably comprise at least about 200, 250,300, 350, 400, 410, 420, 430, 435 or 440 base pairs.

The invention further pertains to nucleic acid molecules for use asprobes/primer or antisense molecules (i.e. non-coding nucleic acidmolecules), which can comprise at least about 6, 12, 20, 30, 50, 100,125, 150 or 200 nucleotides or base pairs. Yet other preferred nucleicacids of the invention comprise at least about 300, at least about 350,at least about 400, at least about 450, at least about 500, or at leastabout 600 nucleotides of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43 or 45. In some embodiments, the nucleic acids of theinvention correspond to a 5′ portion of nucleic acid sequence SEQ ID NO:1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45. For example, anucleic acid of the invention can correspond to a portion of aboutnucleotide 1 to about nucleotide 2000 of nucleic acid sequence SEQ IDNO: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45.

Preferred nucleic acids for use as a probe according to the methods ofthe invention include nucleic acids comprising a nucleotide sequencehaving at least about 6, preferably at least about 9, more preferably atleast about 12 and even more preferably at least about 15 consecutivenucleotides from SEQ ID NOs: Nos:1, 3, 24, 26, 27, 29, 31, 33, 35, 37,39, 41, 43 or 45, or from a portion thereof. In a preferred embodiment,the portion corresponds to about nucleotide 1 to about nucleotide 2060of SEQ ID NOs: Nos:1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or45. Alternatively a portion can be a nucleotide sequence encoding aconserved motif of hDelta3 or mDelta3 protein. Alternatively, theportion can be a nucleotide sequence located between nucleic acidsequences encoding conserved motifs of a human or mouse Delta3 protein.

The invention further provides for a combination of at least two nucleicacids corresponding to at least a portion of SEQ ID NOs: Nos:1, 3, 24,26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or a homolog thereof.Accordingly, in one embodiment, the invention provides a combination oftwo nucleic acids of at least about 6, preferably at least about 9, morepreferably at least about 12 and even more preferably at least about 15consecutive nucleotides from SEQ ID NOs: Nos:1, 3, 24, 26, 27, 29, 31,33, 35, 37, 39, 41, 43 or 45, or from a portion thereof. In a preferredembodiment, at least one of the nucleic acids is labeled.

Another aspect of the invention provides a nucleic acid which hybridizesunder stringent conditions to a nucleic acid represented by one of SEQID Nos:1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45. In yetanother embodiment, a naturally occurring Delta3 nucleic acid of theinvention, e.g., SEQ ID NO: 1, 3, 24 or 26 hybridizes under highstringency conditions to a species variant of Delta3, such as a speciesvariant shown in any one of SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41,43 or 45. In yet another embodiment, a Delta3 nucleic acid, e.g., SEQ IDNO: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45 hybridizesunder high stringency conditions to a representative mammalian Delta3.

Preferred nucleic acids have a sequence at least about 75% identical andmore preferably at least about 80% and even more preferably at leastabout 85% identical with a nucleic acid sequence of a Delta3 gene, suchas a human Delta3 gene or a mouse Delta3 gene, e.g., such as a sequenceshown in one of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39,41, 43 or 45. Nucleic acids at least about 90%, more preferably at leastabout 95%, and most preferably at least about 98-99% homologous with anucleic sequence represented in one of SEQ ID NOs: 1, 3, 24, 26, 27, 29,31, 33, 35, 37, 39, 41, 43 or 45 are of course also within the scope ofthe invention. In preferred embodiments, the nucleic acid is a humanDelta3 gene and in particularly preferred embodiments, includes all or aportion of the nucleotide sequence corresponding to the coding region ofone of SEQ ID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or45.

In addition to naturally-occurring allelic variants of the FTHMA-070,T85, Tango77, T129 or A259 sequence that may exist in the population,the skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequence of SEQ ID NO:53, SEQID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:76, SEQ ID NO:80, 137, 139, 145, 146, 163, 164 or the cDNA of ATCC98807, thereby leading to changes in the amino acid sequence of theencoded FTHMA-070, T85, Tango77, T129 or A259 protein, without alteringthe functional ability of the FTHMA-070, T85, Tango77, T129 or A259protein. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of FTHMA-070, T85, Tango77, T129 or A259 (e.g.,the sequence of SEQ ID NO:54, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77,SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:138, SEQ ID NO:147or SEQ ID NO:165) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the FTHMA-070,T85, Tango77, T129 or A259 proteins of various species are predicted tobe particularly unamenable to alteration.

For example, preferred T85 proteins of the present invention, contain atleast one fibronectin I11 or Ig superfamily domain. Such conserveddomains are less likely to be amenable to mutation. Other amino acidresidues, however, (e.g., those that are not conserved or onlysemi-conserved among T85 of various species) may not be essential foractivity and thus are likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding FTHMA-070, T85, Tango77, T129 or A259 proteins thatcontain changes in amino acid residues that are not essential foractivity. Such FTHMA-070, T85, Tango77, T129 or A259 proteins differ inamino acid sequence from SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:72, SEQID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:138, SEQ ID NO:147 or SEQ ID NO:165, respectively, yet retainbiological activity.

For example, preferred T129 proteins of the present invention, containat least one TNFR/NGFR cysteine rich domain. Such conserved domains areless likely to be amenable to mutation. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conservedamong T129 of various species) may not be essential for activity andthus are likely to be amenable to alteration.

In one embodiment, the isolated nucleic acid molecule includes anucleotide sequence encoding a protein that includes an amino acidsequence that is at least about 45% identical, 65%, 75%, 85%, 95%, or98% identical to the amino acid sequence of SEQ ID NO:72, SEQ ID NO:75,SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:138,SEQ ID NO:147 or SEQ ID NO:165.

An isolated nucleic acid molecule encoding a FTHMA-070, T85, Tango77,T129 or A259 protein having a sequence which differs from that of SEQ IDNO:54, SEQ ID NO:58, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:83, SEQ ID NO:138, SEQ IDNO:147 or SEQ ID NO:165, respectively, can be created by introducing oneor more nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:80, SEQ IDNO:137, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:163 orSEQ ID NO:164 or the cDNA of ATCC 98807 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in FTHMA-070, T85, Tango77, T129 or A259 ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, mutations can be introduced randomly alongall or part of a FTHMA-070, T85, Tango77, T129 or A259 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for FTHMA-070, T85, Tango77, T129 or A259 biological activityto identify mutants that retain activity. Following mutagenesis, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

In a preferred embodiment, a mutant FTHMA-070 or T85 protein can beassayed for the ability to form protein:protein interactions with otherproteins.

In a preferred embodiment, a mutant Tango-77 protein can be assayed for:(1) the ability to form protein:protein interactions with proteins inthe Tango-77 signalling pathway; (2) the ability to bind a Tango-77ligand or receptor; or (3) the ability to bind to an intracellulartarget protein or (4) the ability to interact with a protein involved ininflammation or (5) the ability to bind the IL-1 receptor. In yetanother preferred embodiment, a mutant Tango-77 can be assayed for theability to modulate inflammation, asthma, autoimmune diseases, andsepsis.

In a preferred embodiment, a mutant A259 polypeptide that is a variantof a A259 polypeptide of the invention can be assayed for: (1) theability to form protein:protein interactions with proteins in asignaling pathway of the polypeptide of the invention; (2) the abilityto bind a ligand of the polypeptide of the invention; or (3) the abilityto bind to an intracellular target protein of the polypeptide of theinvention. In yet another preferred embodiment, the mutant polypeptidecan be assayed for the ability to modulate cellular proliferation,cellular migration or chemotaxis, or cellular differentiation.

In addition to naturally-occurring allelic variants of the SPOILsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:104, SEQID NO:112, the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the DNA insert of the plasmid deposited withATCC as Accession Number 98984, thereby leading to changes in the aminoacid sequence of the encoded SPOIL proteins, without altering thefunctional ability of the SPOIL proteins. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:89, SEQ IDNO:101, SEQ ID NO:104, SEQ ID NO:112, the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the DNA insert of theplasmid deposited with ATCC as Accession Number 98984. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of SPOIL (e.g., the sequence of SEQ ID NO:90, SEQ ID NO:102,SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98883, orthe amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98984) without altering thebiological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues ofSPOIL that are conserved among the human and murine family numbers ofthis invention, (as indicated by the alignment and comparison of theamino acid sequences of SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQID NO:113) are predicted to be essential in SPOIL and, thus are notlikely to be amenable to alteration. Table III further sets forthconserved amino residues among SPOIL proteins which are predicted to beunamenable to alteration. Furthermore, amino acid residues that areconserved among the SPOIL proteins of the present invention, and theIL-1ra protein (as indicated by the alignment presented in FIG. 7) arepredicted to be unamenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding SPOIL proteins that contain changes in amino acidresidues that are not essential for activity. Such SPOIL proteins differin amino acid sequence from SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105,SEQ ID NO:113, the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, yet retain biological activity. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 60% identical to the amino acid sequence of SEQID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 65-70% identical to SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105,SEQ ID NO:113, the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, more preferably at least about 75-80% identicalto SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, the aminoacid sequence encoded by the DNA insert of the plasmid deposited withATCC as Accession Number 98883, or the amino acid sequence encoded bythe DNA insert of the plasmid deposited with ATCC as Accession Number98984, even more preferably at least about 85-90% identical to SEQ IDNO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98984, andmost preferably at least about 95% identical to SEQ ID NO:90, SEQ IDNO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encoded bythe DNA insert of the plasmid deposited with ATCC as Accession Number98883, or the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98984.

An isolated nucleic acid molecule encoding a SPOIL protein homologous tothe protein of SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ IDNO:113, the amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113,the DNA insert of the plasmid deposited with ATCC as Accession Number98883, or the DNA insert of the plasmid deposited with ATCC as AccessionNumber 98984, such that one or more amino acid substitutions, additionsor deletions are introduced into the encoded protein. Mutations can beintroduced into SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:104, SEQ IDNO:112, the DNA insert of the plasmid deposited with ATCC as AccessionNumber 98883, or the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a SPOIL coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forSPOIL biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:104, SEQID NO:112, the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the DNA insert of the plasmid deposited withATCC as Accession Number 98984, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant SPOIL-I protein can be assayed for(1) the ability to modulate IL-1 signal transduction, either in vitro orin vivo; (2) modulate IL-1 stimulated cell development ordifferentiation, either in vitro or in vivo; and (3) modulate IL-1stimulated cellular proliferation, either in vitro or in vivo. In yetanother preferred embodiment, a mutant SPOIL can be assayed for abilityto 1) modulate cellular signal transduction; 2) regulate cellularproliferation; 3) regulate cellular differentiation; 4) modulate a cellinvolved in immune response; and 5) modulate a cell involved in bonemetabolism (e.g. osteoblast or osteoclasts).

Moreover, nucleic acid molecules encoding other NEOKINE family members(e.g., NEOKINE-2), and thus which have a nucleotide sequence whichdiffers from the NEOKINE-1 sequences of SEQ ID NO:115, SEQ ID NO:118,SEQ ID NO:121, SEQ ID NO:124, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98751 areintended to be within the scope of the invention. For example, aNEOKINE-2 cDNA can be identified based on the nucleotide sequence ofhuman NEOKINE-1. Moreover, nucleic acid molecules encoding NEOKINEproteins from different species, and thus which have a nucleotidesequence which differs from the NEOKINE sequences of SEQ ID NO:115, SEQID NO:118, SEQ ID NO:121, SEQ ID NO:124, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number98751 are intended to be within the scope of the invention. For example,a Xenopus NEOKINE cDNA can be identified based on the nucleotidesequence of a human NEOKINE.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the NEOKINE cDNAs of the invention can be isolated basedon their homology to the NEOKINE nucleic acids disclosed herein usingthe cDNAs disclosed herein, or a portion thereof, as a hybridizationprobe according to standard hybridization techniques under stringenthybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQID NO:124, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98751. In other embodiment, thenucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 70%, more preferably at leastabout 80%, even more preferably at least about 85% or 90% homologous toeach other typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C., preferably 55° C., and more preferably 60°C. or 65° C. Preferably, an isolated nucleic acid molecule of theinvention that hybridizes under stringent conditions to the sequence ofSEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, correspondsto a naturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

In addition to naturally-occurring allelic variants of the NEOKINEsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQID NO:124, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98751, thereby leading tochanges in the amino acid sequence of the encoded NEOKINE proteins,without altering the functional ability of the NEOKINE proteins. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98751. A “non-essential” amino acid residue is aresidue that can be altered from the wild-type sequence of NEOKINE(e.g., the sequence of SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, orSEQ ID NO:125) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the NEOKINEproteins of the present invention, are predicted to be particularlyunamenable to alteration (e.g., the four conserved cycteines). Moreover,amino acid residues that are defined by the NEOKINE CXC motif areparticularly unamenable to alteration. Furthermore, additional aminoacid residues that are conserved between the NEOKINE proteins of thepresent invention as depicted in FIG. 8 are not likely to be amenable toalteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding NEOKINE proteins that contain changes in amino acidresidues that are not essential for activity. Such NEOKINE proteinsdiffer in amino acid sequence from SEQ ID NO:116, SEQ ID NO:119, SEQ IDNO:122, or SEQ ID NO:125 yet retain biological activity. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 60% homologous to the amino acid sequence of SEQID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125. Preferably,the protein encoded by the nucleic acid molecule is at least about65-70% homologous to SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQID NO:125, more preferably at least about 75-80% homologous to SEQ IDNO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125, even morepreferably at least about 85-90% homologous to SEQ ID NO:116, SEQ IDNO:119, SEQ ID NO:122, or SEQ ID NO:125, and most preferably at leastabout 95% homologous to SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, orSEQ ID NO:125.

An isolated nucleic acid molecule encoding a NEOKINE protein homologousto the protein of SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ IDNO:125 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98751, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO:115, SEQ ID NO:118,SEQ ID NO:121, SEQ ID NO:124, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98751 bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Thus, a predicted nonessential amino acid residue in aNEOKINE protein is preferably replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a NEOKINEcoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for NEOKINE biological activity to identifymutants that retain activity. Following mutagenesis of SEQ ID NO:115,SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98751, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

In a preferred embodiment, a mutant NEOKINE protein can be assayed for(1) modulation of cellular signal transduction, either in vitro or invivo; (2) regulation of gene transcription in a cell expressing aNEOKINE receptor (e.g., RDC1) or receptor which is specific for anotherchemokine; (3) regulation of gene transcription in a cell expressing aNEOKINE receptor or receptor which is specific for another chemokine,wherein said cell is involved in angiogenesis or inflammation; (4)regulation of angiogenesis; (5) regulation of angiogenesis, wherein saidregulation comprises inhibibition of angiogenesis; (6) regulation ofangiogenesis, wherein said regulation comprises maintenance ofangiostasis; (7) regulation of inflammation; and (8) regulation ofinflammation, wherein said regulation comprises inhibition ofchemoattraction (e.g., neutrophil chemoattraction).

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga polypeptide of the invention, e.g., complementary to the coding strandof a double-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can be antisense to all or part of a Delta3, FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 non-coding region ofthe coding strand of a nucleotide sequence encoding a Delta3, FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 polypeptide of theinvention. The non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences which flank the coding region and are nottranslated into amino acids.

Given the coding strand sequences encoding FTHMA-070 or T85 disclosedherein, antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof FTHMA-070 or T85 mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof FTHMA-070 or T85 mRNA.

Given the coding strand sequences encoding Tango-77 disclosed herein(e.g., SEQ ID NO:73, SEQ ID NO:75, or SEQ ID NO:78), antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of Tango-77 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of Tango-77 mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of Tango-77 mRNA, e.g., an oligonucleotidehaving the sequence

5′-TGCAACTTTTACAGGAAACAC-3′ (SEQ ID NO: 193) or5′-CCTCACTTTTACCCGAGACTC-3′ (SEQ ID NO: 194) or5′-GACGGGTGGTACTTAAAACAA-3′. (SEQ ID NO: 195)

Given the coding strand sequences encoding SPOIL disclosed herein (e.g.,SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:112, the DNAinsert of the plasmid deposited with ATCC as Accession Number 98883, orthe DNA insert of the plasmid deposited with ATCC as Accession Number98984), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof SPOIL mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding region of SPOIL mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of SPOIL mRNA.

Given the coding strand sequences encoding NEOKINE disclosed herein(e.g., SEQ ID NO:117, SEQ ID NO:120, or SEQ ID NO:123), antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire coding region of NEOKINE mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of NEOKINE mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of NEOKINE mRNA.

Given the coding strand sequences encoding T129 disclosed herein (e.g.,SEQ ID NO:137 or SEQ ID NO:139), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of T129 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of T129 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of T129 mRNA, e.g., an oligonucleotide having thesequence

CTGGTGGTCCCCGGACTCCTACTTCGGTT (SEQ ID NO: 143) or GACTCCTACTTCGGTTCAGA.(SEQ ID NO: 144)

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleicacid of the invention can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally-occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered, individually or in combination (that is two, three, four,or more different antisense molecules), to a subject or generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding a selected polypeptide of the invention to therebyinhibit expression, e.g., by inhibiting transcription and/ortranslation. The hybridization can be by conventional nucleotidecomplementarity to form a stable duplex, or, for example, in the case ofan antisense nucleic acid molecule which binds to DNA duplexes, throughspecific interactions in the major groove of the double helix. Anexample of a route of administration of antisense nucleic acid moleculesof the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a polypeptide of theinvention can be designed based upon the nucleotide sequence of a cDNAdisclosed herein. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in a Cechet al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, an mRNA encoding a polypeptide of the invention can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, expression of a polypeptide of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23; Perry-O'Keefeet al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.

PNAs of Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129and A259 can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23; or asprobes or primers for DNA sequence and hybridization (Hyrup et al.(1996) Bioorganic & Medicinal Chemistry 4(1): 5-23; Perry-O'Keefe et al.(1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, PNAs of Delta3, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 and A259 can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras can be generated which may combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNAse H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup et al. (1996)Bioorganic & Medicinal Chemistry 4(1): 5-23). The synthesis of PNA-DNAchimeras can be performed as described in Hyrup et al. (1996) Bioorganic& Medicinal Chemistry 4(1): 5-23, and Finn et al. (1996) Nucleic AcidsRes. 24(17):3357-63. For example, a DNA chain can be synthesized on asolid support using standard phosphoramidite coupling chemistry andmodified nucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al. (1989)Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser et al. (1975) Bioorganic Med.Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication NO: WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication NO: WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID Nos: 1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43 or 45 due to degeneracy in the genetic code are alsowithin the scope of the invention. Such nucleic acids encodefunctionally equivalent peptides (i.e., a peptide having a biologicalactivity of a Delta3 polypeptide) but differ from the sequence shown inthe sequence listing due to degeneracy in the genetic code. For example,a number of amino acids are designated by more than one triplet. Codonsthat specify the same amino acid, or synonyms (for example, CAU and CACeach encode histidine) may result in “silent” mutations which do notaffect the amino acid sequence of a Delta3 polypeptide. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequences of the subject Delta3 polypeptides will exist. Oneskilled in the art will appreciate that these variations in one or morenucleotides (e.g., up to about 3-5% of the nucleotides) of the nucleicacids encoding polypeptides having an activity of a Delta3 polypeptidemay exist among individuals of a given species due to natural allelicvariation.

As indicated by the examples set out below, Delta3 protein-encodingnucleic acids can be obtained from mRNA present in any of a number ofeukaryotic cells. It should also be possible to obtain nucleic acidsencoding Delta3 polypeptides of the present invention from genomic DNAfrom both adults and embryos. For example, a gene encoding a Delta3protein can be cloned from either a cDNA or a genomic library inaccordance with protocols described herein, as well as those generallyknown to persons skilled in the art. Examples of tissues and/orlibraries suitable for isolation of the subject nucleic acids includeendothelial cell libraries, among others. A cDNA encoding a Delta3protein can be obtained by isolating total mRNA from a cell, e.g., avertebrate cell, a mammalian cell, or a human cell, including embryoniccells. Double stranded cDNAs can then be prepared from the total mRNA,and subsequently inserted into a suitable plasmid or bacteriophagevector using any one of a number of known techniques. The gene encodinga Delta3 protein can also be cloned using established polymerase chainreaction techniques in accordance with the nucleotide sequenceinformation provided by the invention. The nucleic acid of the inventioncan be DNA or RNA. A preferred nucleic acid is a cDNA represented by asequence selected from the group consisting of SEQ ID NOs: 1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45.

Delta3 Vectors

This invention also provides expression vectors containing a nucleicacid encoding a Delta3 polypeptide, operably linked to at least onetranscriptional regulatory sequence. “Operably linked” is intended tomean that the nucleotide sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe subject Delta3 proteins. Accordingly, the term “transcriptionalregulatory sequence” includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). In one embodiment, the expression vectorincludes a recombinant gene encoding a peptide having an agonisticactivity of a subject Delta3 polypeptide, or alternatively, encoding apeptide which is an antagonistic form of the Delta3 protein. Suchexpression vectors can be used to transfect cells and thereby producepolypeptides, including fusion proteins, encoded by nucleic acids asdescribed herein. Moreover, the gene constructs of the present inventioncan also be used as a part of a gene therapy protocol to deliver nucleicacids encoding either an agonistic or antagonistic form of one of thesubject Delta3 proteins. Thus, another aspect of the invention featuresexpression vectors for in vivo or in vitro transfection and expressionof a Delta3 polypeptide in particular cell types so as to reconstitutethe function of, or alternatively, abrogate the function ofDelta-induced signaling in a tissue. This could be desirable, forexample, when the naturally-occurring form of the protein is expressedinappropriately; or to deliver a form of the protein which altersdifferentiation of tissue. Expression vectors may also be employed toinhibit neoplastic transformation.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a subjectDelta3 polypeptide in the tissue of an animal. Most nonviral methods ofgene transfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral targeting means of the present invention rely onendocytic pathways for the uptake of the subject Delta3 polypeptide geneby the targeted cell. Exemplary targeting means of this type includeliposomal derived systems, polylysine conjugates, and artificial viralenvelopes.

Delta3 Probes and Primers

Moreover, the nucleotide sequences determined from the cloning ofhDelta3 genes will further allow for the generation of probes andprimers designed for use in identifying and/or cloning Delta3 homologsin other cell types, e.g., from other tissues, as well as Delta3homologs from other mammalian organisms. Probes and primers of theinvention can also be used to determine the identity of a Delta3 alleleand/or the presence or absence of one or more mutations in a Delta3 geneof a subject. In a preferred embodiment, a probe or primer of theinvention can be used to determine whether a subject has or is at riskof developing a disease or condition associated with a specific Delta3allele, such as an allele carrying a mutation.

In a preferred embodiment, the present invention also provides aprobe/primer comprising a substantially purified oligonucleotide, whicholigonucleotide comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably about 40, 50 or 75 consecutive nucleotides ofsense or anti-sense sequence selected from the group consisting of SEQID NO:1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, ornaturally-occurring mutants thereof. For instance, primers based on thenucleic acid represented in SEQ ID NOs:1, 3, 24, 26, 27, 29, 31, 33, 35,37, 39, 41, 43 or 45 can be used in PCR reactions to clone Delta3homologs, e.g., specific Delta3 alleles. Such primers are preferablyselected in a region which does not share significant homology to othergenes, e.g., other Delta genes. Examples of primers of the invention areset forth as SEQ ID NOs:12-15, set forth below:

5′ end primers: (SEQ ID NO: 12; corresponding tonucleotides 356 to 375 of SEQ ID NO: 1) 5′ AGCGCCTCTGGCTGGGCGCT 3′;(SEQ ID NO: 13; corresponding to nucleotides 725 to 744 of SEQ ID NO: 1)5′ CGGCCAGAGGCCTTGCCACC 3′; 3′ end primers:(SEQ ID NO: 14; corresponding to the complement of nucleotides1460 to 1479 of SEQ ID NO: 1) 5′ TTGCGCTCCCGGCTGGAGCC 3′; and(SEQ ID NO: 15; corresponding to the complement of nucleotides1592 to 2611 of SEQ ID NO: 1) 5′ ATGCGGCTTGGACCTCGGTT 3′.

Likewise, probes based on the subject Delta3 sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto and able to be detected, e.g., the label group isselected from amongst radioisotopes, fluorescent compounds, enzymes, andenzyme co-factors.

As discussed in more detail below, such probes can also be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress a Delta3 protein, such as by measuring a level of aDelta-encoding nucleic acid in a sample of cells from a patient; e.g.,detecting Delta3 mRNA levels or determining whether a genomic Delta3gene has been mutated or deleted. Briefly, nucleotide probes can begenerated from the subject Delta3 genes which facilitate histologicalscreening of intact tissue and tissue samples for the presence (orabsence) of Delta-encoding transcripts. Similar to the diagnostic usesof anti-Delta3 antibodies, the use of probes directed to Delta3messages, or to genomic Delta3 sequences, can be used for bothpredictive and therapeutic evaluation of allelic mutations which mightbe manifest in, for example, neoplastic or hyperplastic disorders (e.g.,unwanted cell growth) or abnormal differentiation of tissue. Used inconjunction with immunoassays as described herein, the oligonucleotideprobes can help facilitate the determination of the molecular basis fora developmental disorder which may involve some abnormality associatedwith expression (or lack thereof) of a Delta3 protein. For instance,variation in polypeptide synthesis can be differentiated from a mutationin a coding sequence.

Also within the scope of the invention are kits for determining whethera subject is at risk of developing a disease or condition caused by orcontributed by an aberrant Delta3 activity and/or which is associatedwith one or more specific Delta3 alleles. In a preferred embodiment, thekit can be used for determining whether a subject is at risk ofdeveloping a neurological disease or disorder, e.g., a peripheralneuropathy, e.g., ACCPN.

Delta3 Antisense, Ribozyme and Triplex Techniques

One aspect of the invention relates to the use of the isolated nucleicacid in “antisense” therapy. As used herein, “antisense” therapy refersto administration or in situ generation of oligonucleotide molecules ortheir derivatives which specifically hybridize (e.g., bind) undercellular conditions, with the cellular mRNA and/or genomic DNA encodingone or more of the subject Delta3 proteins so as to inhibit expressionof that protein, e.g., by inhibiting transcription and/or translation.The binding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix. In general,“antisense” therapy refers to the range of techniques generally employedin the art, and includes any therapy which relies on specific binding tooligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a Delta3 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of aDelta3 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by Van der Krol etal. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668.

With respect to antisense DNA, oligodeoxyribonucleotides derived fromthe translation initiation site, i.e., the ATG codon which encodes thefirst methionine of the cDNA, e.g., between the −10 and +10 regions ofthe Delta3 nucleotide sequence of interest, are preferred. Preferredantisense molecules of the invention are from nucleotides 328 to 348 ofSEQ ID NO:1 or nucleotides 38 to 58 of SEQ ID NO:24. Non-limitingexamples of preferred human and mouse antisense primers are shown below:

(SEQ ID NO: 16) 5′ TGCCGCCATCCCTCGGGGCGT 3′(complement to nucleotides 326-346 of SEQ ID NO: 1) (SEQ ID NO: 17) 5′GGACGCTGCCGCCATCCCCT 3′(complement to nucleotides 333-352 of SEQ ID NO: 1) (SEQ ID NO: 18) 5′GGACGCTGCCGCCATCCCCTCGGGGCGT 3′(complement to nucleotides 326-352 of SEQ ID NO: 1) (SEQ ID NO: 47) 5′CTCCGGGACGCAGGCGTCATCCCT 3′(complement to nucleotides 38-58 of SEQ ID NO: 24) (SEQ ID NO: 48) 5′ACAGGCGCTCCGGGACGCAGGCGTCATCC 3′(complement to nucleotides 40-65 of SEQ ID NO: 24)

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to Delta3 mRNA. The antisenseoligonucleotides will bind to the Delta3 mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. A sequence “complementary” to a portion of an RNA, as referredto herein, means a sequence having sufficient complementarity to be ableto hybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently been shown to be effective atinhibiting translation of mRNAs as well. (Wagner, R. (1994) Nature372:333). Therefore, oligonucleotides complementary to either the 5′ or3′ untranslated, non-coding regions of a Delta3 gene could be used in anantisense approach to inhibit translation of endogenous Delta3 mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with theinvention. Whether designed to hybridize to the 5′, 3′ or coding regionof Delta3 mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In certain embodiments, theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides, or at least 50 nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. It is preferred that thesestudies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication NO: WO 88/09810, Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication NO: WO 89/10134, Apr.25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol etal. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g.,Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

The anti sense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.(1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleic acids complementary to the coding regionsequence could be used, those complementary to the transcribeduntranslated region are preferred. Antisense nucleic acids overlappingthe site of initiation of translation are even more preferred. Forexample, antisense oligonucleotides as set forth below can be utilizedin accordance with the invention.

5′ TCAATCTGGCTCTGTTCGCG 3′ (SEQ ID NO: 19)(complement to nucleotides 284-303 of SEQ ID NO: 1) 5′CGCTCTCTCCACCCGCGGGCCCTCAA 3′ (SEQ ID NO: 20)(complement to nucleotides 300-325 of SEQ ID NO: 1) 5′GGTGTCCTCTCCACCGGACGCGTGGG 3′ (SEQ ID NO: 49)(complement to nucleotides 6-31 of SEQ ID NO: 24) 5′GTCCTCTCCACCGGACGCGTGG 3′ (SEQ ID NO: 50)(complement to nucleotides 6-28 of SEQ ID NO: 24)

The antisense molecules should be delivered to cells which express theDelta3 in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation on endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous Delta3 transcripts andthereby prevent translation of the Delta3 mRNA. For example, a vectorcan be introduced in vivo such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon (1981) Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al. (1980) Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al. (1982) Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systematically).

Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of one of the Delta3proteins, can be used in the modulation of cellular activity both invivo and for ex vivo tissue cultures.

Furthermore, the anti-sense techniques (e.g., microinjection ofantisense molecules, or transfection with plasmids whose transcripts areanti-sense with regard to a Delta3 mRNA or gene sequence) can be used toinvestigate the role of Delta3 in developmental events, as well as thenormal cellular function of Delta3 in adult tissue. Such techniques canbe utilized in cell culture, but can also be used in the creation oftransgenic animals, as detailed below.

Ribozyme molecules designed to catalytically cleave Delta3 mRNAtranscripts can also be used to prevent translation of mRNA andexpression of Delta3. (See, e.g., PCT International Publication WO94/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by an endonucleolytic cleavage.The composition of ribozyme molecules must include one or more sequencescomplementary to the target gene mRNA, and must include the well knowncatalytic sequence responsible for mRNA cleavage. For this sequence, seeU.S. Pat. No. 5,093,246. As such within the scope of the invention areengineered hammerhead motif ribozyme molecules that specifically andefficiently catalyze endonucleolytic cleavage of RNA sequences encodingDelta3 proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the molecule of interest for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatesequences may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

While ribozymes that cleave mRNA at site specific recognition sequencescan be used to destroy Delta3 mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach (1988) Nature 334:585-591. There arehundreds of potential hammerhead ribozyme cleavage sites within thenucleotide sequence of human Delta3 cDNA. Preferably the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the Delta3 mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al. (1984) Science, 224:574-578; Zaug andCech (1986) Science, 231:470-475; Zaug, et al. (1986) Nature324:429-433; PCT Publication WO 88/04300; Been & Cech (1986) Cell47:207-216). The Cech-type ribozymes have an eight base pair active sitewhich hybridizes to a target RNA sequence whereafter cleavage of thetarget RNA takes place. The invention encompasses those Cech-typeribozymes which target eight base-pair active site sequences that arepresent in Delta3.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express the Delta3 in vivo e.g.,hypothalamus and/or the choroid plexus. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive pol III or pol II promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous Delta3 messages and inhibit translation. Because ribozymesunlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

Endogenous Delta3 gene expression can also be reduced by inactivating or“knocking out” the Delta3 gene or its promoter using targeted homologousrecombination. (e.g., see Smithies et al. (1985) Nature 317:230-234;Thomas & Capecchi (1987) Cell 51:503-512; Thompson et al. (1989) Cell5:313-321). For example, a mutant, non-functional Delta3 (or acompletely unrelated DNA sequence) flanked by DNA homologous to theendogenous Delta3 gene (either the coding regions or regulatory regionsof the Delta3 gene) can be used, with or without a selectable markerand/or a negative selectable marker, to transfect cells that expressDelta3 in vivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the Delta3 gene. Suchapproaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive Delta3 (e.g., see Thomas & Capecchi(1987) and Thompson (1989), supra). However this approach can be adaptedfor use in humans provided the recombinant DNA constructs are directlyadministered or targeted to the required site in vivo using appropriateviral vectors, e.g., herpes virus vectors.

Alternatively, endogenous Delta3 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the Delta3 gene (i.e., the Delta3 promoter and/or enhancers)to form triple helical structures that prevent transcription of theDelta3 gene in target cells in the body. (See generally, Helene, C.(1991) Anticancer Drug Des. 6(6):569-84; Helene, C., et al. (1992) Ann.N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′,3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligo-deoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 and A259Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding Delta3,FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operatively linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 proteins, mutant forms ofDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259,fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 in prokaryotic or eukaryotic cells, e.g., bacterialcells such as E. coli, insect cells (using baculovirus expressionvectors) yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a 17 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., Sf 9 cells) include the pAcseries (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal. (1989) Cold Spring Harbor Laboratory (supra).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Camper andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes See Weintraub et al., Reviews—Trends inGenetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced.

Another aspect of the invention pertains to host cells into which aSPOIL nucleic acid molecule of the invention is introduced, e.g., aSPOIL nucleic acid molecule within a recombinant expression vector or aSPOIL nucleic acid molecule in a form suitable for homologousrecombination in the genome of a host cell (e.g., a SPOIL nucleic acidmolecule which includes SPOIL nucleotide sequences and additional 5′ and3′ flanking sequences necessary for homologous recombination).

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (supra), andother laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

In another embodiment, the expression characteristics of an endogenous(e.g., Delta3) nucleic acid within a cell, cell line or microorganismmay be modified by inserting a DNA regulatory element heterologous tothe endogenous gene of interest into the genome of a cell, stable cellline or cloned microorganism such that the inserted regulatory elementis operatively linked with the endogenous gene (e.g., Delta3) andcontrols, modulates or activates the endogenous gene. For example,endogenous Delta3 which is normally “transcriptionally silent”, i.e.,Delta3 which is normally not expressed, or is expressed only at very lowlevels in a cell line or microorganism, may be activated by inserting aregulatory element which is capable of promoting the expression of anormally expressed gene product in that cell line or microorganism.Alternatively, transcriptionally silent, endogenous Delta3 may beactivated by insertion of a promiscuous regulatory element that worksacross cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked withand activates expression of endogenous Delta3, using techniques, such astargeted homologous recombination, which are well known to those ofskill in the art, and described e.g., in Chappel, U.S. Pat. No.5,272,071, incorporated herein by reference in its entirety; PCTpublication NO: WO 91/06667, published May 16, 1991.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) Delta3,FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein.Accordingly, the invention further provides methods for producingDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 has been introduced) in asuitable medium such that Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein is produced. In another embodiment,the method further comprises isolating Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 from the medium or the hostcell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous Delta3,FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequenceshave been introduced into their genome or homologous recombinant animalsin which endogenous Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 sequences have been altered. Such animals are usefulfor studying the function and/or activity of Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 and for identifying and/orevaluating modulators of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 activity. As used herein, a “transgenic animal” is anon-human animal, preferably a mammal, more preferably a rodent such asa rat or mouse, in which one or more of the cells of the animal includesa transgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, an “homologous recombinant animal” is anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The Delta3,FTHMA-070, T85 or A259 cDNA sequence can be introduced as a transgeneinto the genome of a non-human animal. The Tango-77 cDNA sequence e.g.,that of (SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76; SEQ ID NO:80 or thecDNA of ATCC 98807) can be introduced as a transgene into the genome ofa non-human animal. The human SPOIL cDNA sequence of SEQ ID NO:101, SEQID NO:104, the DNA insert of the plasmid deposited with ATCC asAccession Number 98883, or the DNA insert of the plasmid deposited withATCC as Accession Number 98984, can be introduced as a transgene intothe genome of a non-human animal. The NEOKINE-1 cDNA sequence of SEQ IDNO:115 can be introduced as a transgene into the genome of a non-humananimal. Alternatively, a nonhuman homologue of a human NEOKINE-1 gene,such as a mouse NEOKINE-1 gene (SEQ ID NO:118), a rat NEOKINE-1 gene(SEQ ID NO:121), or a macaque NEOKINE cDNA (SEQ ID NO:124), can be usedas a transgene. The T129 cDNA sequence e.g., that of (SEQ ID NO:137 orSEQ ID NO:139) can be introduced as a transgene into the genome of anon-human animal.

Alternatively, a nonhuman homologue of the human Delta3, FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene, such as a mouseDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259gene, can be isolated based on hybridization to the human Delta3,FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 cDNA andused as a transgene. Intronic sequences and polyadenylation signals canalso be included in the transgene to increase the efficiency ofexpression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the Delta3, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 transgene to direct expression ofDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No.4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similarmethods are used for production of other transgenic animals. Atransgenic founder animal can be identified based upon the presence ofthe Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 transgene in its genome and/or expression of Delta3, FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 mRNA in tissues orcells of the animals. A transgenic founder animal can then be used tobreed additional animals carrying the transgene. Moreover, transgenicanimals carrying a transgene encoding Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 can further be bred to othertransgenic animals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a Delta3, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 gene (e.g., a human or a non-humanhomolog of the Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene, e.g., a murine Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene) into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 gene. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenousDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259gene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thevector can be designed such that, upon homologous recombination, theendogenous Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 gene is mutated or otherwise altered but still encodesfunctional protein (e.g., the upstream regulatory region can be alteredto thereby alter the expression of the endogenous Delta3, FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein). In thehomologous recombination vector, the altered portion of the Delta3,FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene isflanked at its 5′ and 3′ ends by additional nucleic acid of the Delta3,FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene toallow for homologous recombination to occur between the exogenousDelta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259gene carried by the vector and an endogenous Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene in an embryonic stemcell. The additional flanking Delta3, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 nucleic acid is of sufficient lengthfor successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas and Capecchi (1987)Cell 51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced Delta3, FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene has homologouslyrecombined with the endogenous Delta3, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 gene are selected (see e.g., Li et al.(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.In brief, a cell, e.g., a somatic cell, from the transgenic animal canbe isolated and induced to exit the growth cycle and enter G_(o) phase.The quiescent cell can then be fused, e.g., through the use ofelectrical pulses, to an enucleated oocyte from an animal of the samespecies from which the quiescent cell is isolated. The reconstructedoocyte is then cultured such that it develops to morula or blastocyteand then transferred to pseudopregnant female foster animal. Theoffspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Delta3 Peptides of the Present Invention

The present invention also makes available Delta3 polypeptides which areisolated from, or otherwise substantially free of other cellularproteins, especially other signal transduction factors and/ortranscription factors which may normally be associated with the Delta3polypeptide. In general, polypeptides of the invention exhibit anactivity of a Delta3 protein. The invention provides various forms ofDelta3 proteins, specifically including all of the Delta3 proteinsencoded by a nucleic acid of the invention, as described above.

In one embodiment, a polypeptide of the invention is a polypeptide thatis a variant of a polypeptide of the invention can be assayed for: (1)the ability to form protein:protein interactions with proteins in asignaling pathway of the polypeptide of the invention; (2) the abilityto bind a ligand of the polypeptide of the invention; or (3) the abilityto bind to an intracellular target protein of the polypeptide of theinvention. In yet another preferred embodiment, the mutant polypeptidecan be assayed for the ability to modulate cellular proliferation,cellular migration or chemotaxis, or cellular differentiation.

Full-length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast about 5, 10, 25, 50, 75, 100, 125, 150 amino acids in length arewithin the scope of the present invention. The invention encompasses allproteins encoded by the nucleic acids described in the above sectiondescribing the nucleic acids of the invention.

For example, isolated Delta3 polypeptides can include all or a portionof an amino acid sequences corresponding to a Delta3 polypeptiderepresented in SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or46, or the amino acid sequence encoded by the cDNA of a clone depositedwith the ATCC® as Accession Number 98348. Isolated portions of Delta3proteins can be obtained, for example, by screening peptidesrecombinantly produced from the corresponding fragment of the nucleicacid encoding such peptides. In addition, fragments can be chemicallysynthesized using techniques known in the art such as conventionalMerrifield solid phase f-Moc or t-Boc chemistry. For example, a Delta3polypeptide of the present invention may be divided into fragments ofdesired length with no overlap of the fragments, or preferably dividedinto overlapping fragments of a desired length. The fragments can beproduced (recombinantly or by chemical synthesis) and tested to identifythose peptidyl fragments which can function as either agonists orantagonists of a wild-type (e.g., “authentic”) Delta3 protein.

In one embodiment, the Delta3 polypeptide of the invention has anoverall amino acid sequence similarity or identity of at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or at leastabout 99% with the amino acid sequence SEQ ID NOs: 2, 25, 28, 30, 32,34, 36, 38, 40, 42, 44 or 46, or the amino acid sequence encoded by thecDNA of a clone deposited with the ATCC® as Accession Number 98348. In aparticularly preferred embodiment a Delta3 protein has the amino acidsequence SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, orthe amino acid sequence of the cDNA of a clone deposited with the ATCC®as Accession Number 98348. In other particularly preferred embodiments,the Delta3 protein has a Delta3 activity.

The present invention further pertains to forms of one of the subjectDelta3 polypeptides which are encoded by nucleotide sequences derivedfrom a mammalian organism, and which have amino acid sequencesevolutionarily related to the Delta3 protein represented in SEQ ID NOs:2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acidsequence encoded by the cDNA of a clone deposited with the ATCC® asAccession Number 98348. Such recombinant Delta3 polypeptides can, incertain embodiments, preferably are capable of functioning in one ofeither role of an agonist or antagonist of at least one biologicalactivity of a wild-type (“authentic”) Delta3 protein of the appendedsequence listing. The term “evolutionarily related to”, with respect toamino acid sequences of human Delta3 proteins, refers to bothpolypeptides having amino acid sequences which have arisen naturally,and also to mutational variants of the Delta3 polypeptides which arederived, for example, by combinatorial mutagenesis. Such evolutionarilyderived Delta3 polypeptides preferred by the present invention have aDelta3 activity and are at least 80% homologous and more preferably 85%identical and most preferably 90% identical with the amino acid sequenceof SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or theamino acid sequence encoded by the cDNA of a clone deposited with theATCC® as Accession Number 98348. In a particularly preferred embodiment,a Delta3 protein comprises the amino acid coding sequence of SEQ ID NOs:2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or the amino acidsequence of the cDNA of a clone deposited with the ATCC® as AccessionNumber 98348.

The present invention further pertains to methods of producing thesubject Delta3 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The cells may beharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The recombinant Delta3 polypeptide can beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for such peptide. In a preferred embodiment, the recombinantDelta3 polypeptide is a fusion protein containing a domain whichfacilitates its purification, such as GST fusion protein or poly(His)fusion protein.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide variants of one of thesubject Delta3 polypeptides which function in a limited capacity as oneof either a Delta3 agonist (mimetic) or a Delta3 antagonist, in order topromote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a variant having a limitedfunction, and with fewer side effects relative to treatment withagonists or antagonists which are directed to all of the biologicalactivities of naturally-occurring forms of Delta3 proteins.

Variants and/or mutants of each of the subject Delta3 proteins can begenerated by mutagenesis, such as by discrete point mutation(s), or bytruncation. For instance, mutation can give rise to homologs whichretain substantially the same, or merely a subset, of the biologicalactivity of the Delta3 polypeptide from which it was derived.Alternatively, antagonistic forms of the protein can be generated whichare able to inhibit the function of the naturally-occurring form of theprotein, such as by competitively binding to a downstream or upstreammember of the Delta3 cascade which includes the Delta3 protein. Inaddition, agonistic forms of the protein may be generated which areconstitutively active.

The recombinant Delta3 polypeptides of the present invention alsoinclude homologs of the authentic Delta3 proteins, such as versions ofthose protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter ubiquitination or other enzymatictargeting associated with the protein.

Delta3 polypeptides may also be chemically modified to create Delta3derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of Delta3 proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

Modification of the structure of the subject Delta3 polypeptides can befor such purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo), or post-translational modifications (e.g., toalter phosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the Delta3 polypeptides describedin more detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (See, for example, Biochemistry, 4th ed., Ed. by L. Stryer,WH Freeman and Co.: 1995). Whether a change in the amino acid sequenceof a peptide results in a functional Delta3 homolog (e.g., functional inthe sense that the resulting polypeptide mimics or antagonizes thewild-type form) can be readily determined by assessing the ability ofthe variant peptide to produce a response in cells in a fashion similarto the wild-type protein, or competitively inhibit such a response.Polypeptides in which more than one replacement has taken place canreadily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject Delta3 proteins as well astruncation mutants, and is especially useful for identifying potentialfunctional variant sequences (e.g., homologs). The purpose of screeningsuch combinatorial libraries is to generate, for example, novel Delta3homologs which can act as either agonists or antagonist, oralternatively, possess novel activities all together.

In one embodiment, the variegated library of Delta3 variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential Delta3 sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofDelta3 sequences therein.

There are many ways by which such libraries of potential Delta3 variantscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential Delta3 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierppg. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.11:477. Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for aDelta3 clone in order to generate a variegated population of Delta3fragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of a Delta3 coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of Delta3 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate Delta3 sequences created bycombinatorial mutagenesis techniques.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 1026 molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays. To overcome thisproblem, a new technique has been developed recently, recrusive ensemblemutagenesis (REM), which allows one to avoid the very high proportion ofnon-functional proteins in a random library and simply enhances thefrequency of functional proteins, thus decreasing the complexityrequired to achieve a useful sampling of sequence space. REM is analgorithm which enhances the frequency of functional mutants in alibrary when an appropriate selection or screening method is employed(Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan et al., 1992,Parallel Problem Solving from Nature, 2., In Maenner and Manderick,eds., Elsevir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al.,1993, Protein Engineering 6(3):327-331).

The invention also provides for reduction of the Delta3 proteins togenerate mimetics, e.g., peptide or non-peptide agents, which are ableto bind to a Delta3 protein and/or to disrupt binding of a Delta3polypeptide of the present invention with either upstream or downstreamcomponents of a Delta/Notch signaling cascade, such as binding proteinsor interactors. Thus, such mutagenic techniques as described above arealso useful to map the determinants of the Delta3 proteins whichparticipate in protein-protein interactions involved in, for example,binding of the subject Delta3 polypeptide to proteins which may functionupstream (including both activators and repressors of its activity) orto proteins or nucleic acids which may function downstream of the Delta3polypeptide, whether they are positively or negatively regulated by it,for example, Notch. To illustrate, the critical residues of a subjectDelta3 polypeptide which are involved in molecular recognition of, forexample, the Notch gene product or other component upstream ordownstream of a Delta3 gene can be determined and used to generateDelta-derived peptidomimetics which competitively inhibit binding of theauthentic Delta3 protein with that moiety. By employing, for example,scanning mutagenesis to map the amino acid residues of each of thesubject Delta3 proteins which are involved in binding otherextracellular proteins, peptidomimetic compounds can be generated whichmimic those residues of the Delta3 protein which facilitate theinteraction. Such mimetics may then be used to interfere with the normalfunction of a Delta3 protein. For instance, non-hydrolyzable peptideanalogs of such residues can be generated using benzodiazepine (e.g.,see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

Delta3 Fusion Proteins and Immunogens.

In another embodiment, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide.

In one embodiment, the Delta3 polypeptide is a Delta3-Ig polypeptide.The Delta3-Ig polypeptide can comprise the entire extracellular domainof Delta3, e.g., human Delta3, or a variant thereof. For example, aDelta3-Ig polypeptide can comprise an amino acid sequences from aboutamino acid 1 to about amino acid 529 of SEQ ID NO: 2 or from about aminoacid 1 to about amino acid 530 of SEQ ID NO: 25. Other preferredDelta3-Ig proteins do not comprise a signal peptide and thus, preferablydo not comprise about amino acid 1 to about amino acid 17 or amino acid18 of SEQ ID NO: 2 or 25. Alternatively, a Delta3-Ig fusion protein cancomprise a portion of the extracellular domain of a Delta3 protein or avariant of a portion of the extracellular domain of a Delta3 protein.Preferred portions of the extracellular domain include portions havingat least one motif amino terminal to the transmembrane domain. Forexample a Delta3-Ig fusion protein can comprise at least one EGF-likedomain. A Delta3-Ig fusion protein can further comprise a DSL domain. ADelta3-Ig fusion protein can also further comprise a signal peptide.Delta3-Ig fusion proteins can be prepared as described, e.g., in U.S.Pat. No. 5,434,131.

This type of expression system can be useful under conditions where itis desirable to produce an immunogenic fragment of a Delta3 protein. Forexample, the VP6 capsid protein of rotavirus can be used as animmunologic carrier protein for portions of the Delta3 polypeptide,either in the monomeric form or in the form of a viral particle. Thenucleic acid sequences corresponding to the portion of a subject Delta3protein to which antibodies are to be raised can be incorporated into afusion gene construct which includes coding sequences for a latevaccinia virus structural protein to produce a set of recombinantviruses expressing fusion proteins comprising Delta3 epitopes as part ofthe virion. It has been demonstrated with the use of immunogenic fusionproteins utilizing the Hepatitis B surface antigen fusion proteins thatrecombinant Hepatitis B virions can be utilized in this role as well.Similarly, chimeric constructs coding for fusion proteins containing aportion of a Delta3 protein and the poliovirus capsid protein can becreated to enhance immunogenicity of the set of polypeptide antigens(see, for example, EP Publication No: 0259149; and Evans et al. (1989)Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger etal. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofa Delta3 polypeptide is obtained directly from organo-chemical synthesisof the peptide onto an oligomeric branching lysine core (see, forexample, Posnett et al. (1988) J. Biol. Chem. 263:1719 and Nardelli etal. (1992) J. Immunol. 148:914). Antigenic determinants of Delta3proteins can also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the Delta3 polypeptides of the present invention. For example, Delta3polypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of theDelta3 polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. Proc. Natl. Acad. Sci. USA88:8972). Techniques for making fusion genes are known to those skilledin the art. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Delta3 Antibodies

Another aspect of the invention pertains to an antibody that binds to aDelta3 protein; that is, to antibodies directed against a polypeptide ofthe invention. For example, by using immunogens derived from a Delta3protein, e.g., based on the cDNA sequences, anti-protein/anti-peptideantisera or monoclonal antibodies can be made by standard protocols(See, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, ahamster or rabbit can be immunized with an immunogenic form of thepeptide (e.g., a Delta3 polypeptide or an antigenic fragment which iscapable of eliciting an antibody response, or a fusion protein asdescribed above). Techniques for conferring immunogenicity on a proteinor peptide include conjugation to carriers or other techniques wellknown in the art. An immunogenic portion of a Delta3 protein can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of aDelta3 polypeptide, anti-Delta3 antisera can be obtained and, ifdesired, polyclonal anti-Delta3 antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a Delta3 polypeptideof the present invention and monoclonal antibodies isolated from aculture comprising such hybridoma cells. In one embodiment anti-humanDelta3 antibodies specifically react with the proteins encoded by theDNA of ATCC® Deposit Accession Number 98348.

Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)2 fragments can be generated bytreating antibody with pepsin. The resulting F(ab)2 fragment can betreated to reduce disulfide bridges to produce Fab fragments. Theantibody of the present invention is further intended to includebispecific and chimeric molecules having affinity for a Delta3 proteinconferred by at least one CDR region of the antibody.

Antibodies which specifically bind Delta3 epitopes can also be used inimmunohistochemical staining of tissue samples in order to evaluate theabundance and pattern of expression of each of the subject Delta3polypeptides. Anti-Delta3 antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate Delta3protein levels in tissue as part of a clinical testing procedure. Forinstance, such measurements can be useful in predictive valuations ofthe onset or progression of neurodegenerative, neoplastic orhyperplastic disorders. Likewise, the ability to monitor Delta3 proteinlevels in an individual can allow determination of the efficacy of agiven treatment regimen for an individual afflicted with such adisorder. The level of Delta3 polypeptides may be measured from cells inbodily fluid, such as in samples of cerebral spinal fluid or amnioticfluid, or can be measured in tissue, such as produced by biopsy.Diagnostic assays using anti-Delta3 antibodies can include, for example,immunoassays designed to aid in early diagnosis of a neurodegenerativedisorder, particularly ones which are manifest at birth. Diagnosticassays using anti-Delta3 polypeptide antibodies can also includeimmunoassays designed to aid in early diagnosis and phenotypingneurodegenerative, neoplastic or hyperplastic disorders.

Another application of anti-Delta3 antibodies of the present inventionis in the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and ORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of a Delta3protein, e.g., other orthologs of a particular Delta3 protein or otherparalogs from the same species, can then be detected with antibodies,as, for example, reacting nitrocellulose filters lifted from infectedplates with anti-Delta3 antibodies. Positive phage detected by thisassay can then be isolated from the infected plate. Thus, the presenceof Delta3 homologs can be detected and cloned from other animals, as canalternate isoforms (including splicing variants) from humans.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.Preferred polyclonal antibody compositions are ones that have beenselected for antibodies directed against a polypeptide or polypeptidesof the invention. Particularly preferred polyclonal antibodypreparations are ones that contain only antibodies directed against apolypeptide or polypeptides of the invention. Particularly preferredimmunogen compositions are those that contain no other human proteinssuch as, for example, immunogen compositions made using a non-human hostcell for recombinant expression of a polypeptide of the invention. Insuch a manner, the only human epitope or epitopes recognized by theresulting antibody compositions raised against this immunogen will bepresent as part of a polypeptide or polypeptides of the invention.

The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. Alternatively, antibodiesspecific for a protein or polypeptide of the invention can be selectedfor (e.g., partially purified) or purified by, e.g., affinitychromatography. For example, a recombinantly expressed and purified (orpartially purified) protein of the invention is produced as describedherein, and covalently or non-covalently coupled to a solid support suchas, for example, a chromatography column. The column can then be used toaffinity purify antibodies specific for the proteins of the inventionfrom a sample containing antibodies directed against a large number ofdifferent epitopes, thereby generating a substantially purified antibodycomposition, i.e., one that is substantially free of contaminatingantibodies. By a substantially purified antibody composition is meant,in this context, that the antibody sample contains at most only 30% (bydry weight) of contaminating antibodies directed against epitopes otherthan those on the desired protein or polypeptide of the invention, andpreferably at most 20%, yet more preferably at most 10%, and mostpreferably at most 5% (by dry weight) of the sample is contaminatingantibodies. A purified antibody composition means that at least 99% ofthe antibodies in the composition are directed against the desiredprotein or polypeptide of the invention.

At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing hybridomas is well known (see generally CurrentProtocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons,Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibodyof the invention are detected by screening the hybridoma culturesupernatants for antibodies that bind the polypeptide of interest, e.g.,using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog NO: 27-9400-01; and theStratagene SurfZAP Phage Display Kit, Catalog NO: 240612). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, U.S. Pat. No. 5,223,409; PCT Publication No: WO 92/18619; PCTPublication No: WO 91/17271; PCT Publication No: WO 92/20791; PCTPublication No: WO 92/15679; PCT Publication No: WO 93/01288; PCTPublication No: WO 92/01047; PCT Publication No: WO 92/09690; PCTPublication No: WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarily determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089.) Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNO: WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication NO: WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced, forexample, using transgenic mice which are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA and IgE antibodies. For an overview of this technology for producinghuman antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol.13:65-93). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat.No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; andU.S. Pat. No. 5,545,806. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

An antibody directed against a polypeptide of the invention (e.g.,monoclonal antibody) can be used to isolate the polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, such an antibody can be used to detect the protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the polypeptide. The antibodies can also beused diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Further, an antibody (or fragment thereof) can be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, .alpha.-interferon, .beta.-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, incorporated herein by reference in its entirety.

Accordingly, in one aspect, the invention provides substantiallypurified antibodies or fragment thereof, and human and non-humanantibodies or fragments thereof, which antibodies or fragmentsspecifically bind to a polypeptide comprising an amino acid sequenceselected from the group consisting of: the amino acid sequence of anyone of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, oran amino acid sequence encoded by the cDNA of a clone deposited as ATCC®98348; a fragment of at least 15 amino acid residues of the amino acidsequence of any one of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40,42, 44 or 46, an amino acid sequence which is at least 95% identical tothe amino acid sequence of any one of SEQ ID NOs: 2, 25, 28, 30, 32, 34,36, 38, 40, 42, 44 or 46, wherein the percent identity is determinedusing the ALIGN program of the GCG software package with a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4; andan amino acid sequence which is encoded by a nucleic acid molecule whichhybridizes to the nucleic acid molecule consisting of any one of SEQ IDNos:1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the cDNAof a clone deposited as ATCC® 98348, or a complement thereof, underconditions of hybridization of 6×SSC at 45□C. and washing in 0.2×SSC,0.1% SDS at 65□C. In various embodiments, the substantially purifiedantibodies of the invention, or fragments thereof, can be human,non-human, chimeric and/or humanized antibodies.

In another aspect, the invention provides human and non-human antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of any one of SEQ ID NOs: 2, 25,28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acid sequenceencoded by the cDNA of a clone deposited as ATCC® 98348; a fragment ofat least 15 amino acid residues of the amino acid sequence of any one ofSEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, an aminoacid sequence which is at least 95% identical to the amino acid sequenceof any one of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or46, wherein the percent identity is determined using the ALIGN programof the GCG software package with a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4; and an amino acid sequencewhich is encoded by a nucleic acid molecule which hybridizes to thenucleic acid molecule consisting of any one of SEQ ID Nos:1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the cDNA of a clonedeposited as ATCC® 98348, or a complement thereof, under conditions ofhybridization of 6×SSC at 45□C. and washing in 0.2×SSC, 0.1% SDS at65□C. Such non-human antibodies can be goat, mouse, sheep, horse,chicken, rabbit, or rat antibodies. Alternatively, the non-humanantibodies of the invention can be chimeric and/or humanized antibodies.In addition, the non-human antibodies of the invention can be polyclonalantibodies or monoclonal antibodies.

In still a further aspect, the invention provides monoclonal antibodiesor fragments thereof, which antibodies or fragments specifically bind toa polypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of any one of SEQ ID NOs: 2, 25,28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acid sequenceencoded by the cDNA of a clone deposited as ATCC® 98348; a fragment ofat least 15 amino acid residues of the amino acid sequence of any one ofSEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, an aminoacid sequence which is at least 95% identical to the amino acid sequenceof any one of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or46, wherein the percent identity is determined using the ALIGN programof the GCG software package with a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4; and an amino acid sequencewhich is encoded by a nucleic acid molecule which hybridizes to thenucleic acid molecule consisting of any one of SEQ ID NOs: 1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the cDNA of a clonedeposited as any of ATCC® 98348, or a complement thereof, underconditions of hybridization of 6×SSC at 45□C. and washing in 0.2×SSC,0.1% SDS at 65□C. The monoclonal antibodies can be human, humanized,chimeric and/or non-human antibodies.

The substantially purified antibodies or fragments thereof specificallybind to a signal peptide, a secreted sequence, an extracellular domain,a transmembrane or a cytoplasmic domain cytoplasmic membrane of apolypeptide of the invention. In a particularly preferred embodiment,the substantially purified antibodies or fragments thereof, the humanand non-human antibodies or fragments thereof, and/or the monoclonalantibodies or fragments thereof, of the invention specifically bind to asecreted sequence or an extracellular domain of the amino acid sequenceof SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46.Preferably, the secreted sequence or extracellular domain to which theantibody, or fragment thereof, binds comprises from about amino acids1-529 or 18-529 of SEQ ID NO: 2, or from amino acids 1-530 or 18-530 ofSEQ ID NO: 25.

Any of the antibodies of the invention can be conjugated to atherapeutic moiety or to a detectable substance. Non-limiting examplesof detectable substances that can be conjugated to the antibodies of theinvention are an enzyme, a prosthetic group, a fluorescent material, aluminescent material, a bioluminescent material, and a radioactivematerial.

The invention also provides a kit containing an antibody of theinvention, and instructions for use. In another embodiment, a kitcomprising an antibody of the invention conjugated to a detectablesubstance and instructions for use. Still another aspect of theinvention is a pharmaceutical composition comprising an antibody of theinvention and a pharmaceutically acceptable carrier. In preferredembodiments, the pharmaceutical composition contains an antibody of theinvention, a therapeutic moiety, and a pharmaceutically acceptablecarrier.

Still another aspect of the invention is a method of making an antibodythat binds, that is, is directed against, Delta3, the method comprisingimmunizing a mammal with a polypeptide. The polypeptide used as animmungen comprises an amino acid sequence selected from the groupconsisting of: the amino acid sequence of any one of SEQ ID NOs: 2, 25,28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acid sequenceencoded by the cDNA of a clone deposited as ATCC® 98348; a fragment ofat least 15 amino acid residues of the amino acid sequence of any one ofSEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, an aminoacid sequence which is at least 95% identical to the amino acid sequenceof any one of SEQ ID NOs: 2, 25, 28, 30, 32, 34, 36, 38, 40, 42, 44 or46, wherein the percent identity is determined using the ALIGN programof the GCG software package with a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4; and an amino acid sequencewhich is encoded by a nucleic acid molecule which hybridizes to thenucleic acid molecule consisting of any one of SEQ ID NOs:1, 3, 24, 26,27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or the cDNA of a clonedeposited as ATCC® 98348, or a complement thereof, under conditions ofhybridization of 6×SSC at 45□C. and washing in 0.2×SSC, 0.1% SDS at65□C. After immunization, a sample is collected from the mammal thatcontains an antibody that specifically recognizes Delta3. Preferably,the polypeptide is recombinantly produced using a non-human host cell.Optionally, the antibodies can be further purified from the sample usingtechniques well known to those of skill in the art. The method canfurther comprise producing a monoclonal antibody-producing cell from thecells of the mammal. Optionally, antibodies are collected from theantibody-producing cell.

Isolated FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259Proteins and Anti-FTHMA-070 or Anti-T85 Antibodies

One aspect of the invention pertains to isolated FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 proteins, and biologicallyactive portions thereof, as well as polypeptide fragments suitable foruse as immunogens to raise anti-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antibodies. In one embodiment, nativeFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteinscan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein or polypeptide can besynthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 protein in which the proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein that is substantially free ofcellular material includes preparations of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein having less than about 30%,20%, 10%, or 5% (by dry weight) of non-FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein (also referred to herein as a“contaminating protein”). When the FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein is producedby chemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein have less than about 30%, 20%,10%, 5% (by dry weight) of chemical precursors or non-FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 chemicals.

Biologically active portions of a FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein include peptides comprising amino acidsequences sufficiently identical to or derived from the amino acidsequence of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 protein, which include less amino acids than the full lengthFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteins,and exhibit at least one activity of a FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein. Typically, biologically activeportions comprise a domain or motif with at least one activity of theFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein. Abiologically active portion of a FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Preferredbiologically active polypeptides include one or more identifiedstructural domains. Preferred biologically active polypeptides includeone or more identified T129 structural domains, e.g., TNFR/NGFRcysteine-rich domain (SEQ ID NO:142).

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeFTHMA-070 or T85 protein. Preferred FTHMA-070 or T85 protein has theamino acid sequence shown of SEQ ID NO:54 or SEQ ID NO:58, respectively.Other useful FTHMA-070 or T85 proteins are substantially identical toSEQ ID NO:54 or SEQ ID NO:58, respectively and retain the functionalactivity of the reference protein yet differ in amino acid sequence dueto natural allelic variation or mutagenesis.

Accordingly, a useful FTHMA-070 protein is a protein which includes anamino acid sequence at least about 45%, preferably 55%, 65%, 75%, 85%,95%, or 99% identical to the amino acid sequence of SEQ ID NO:54 andretains the functional activity of the FTHMA-070 protein of SEQ IDNO:54. In a preferred embodiment, the FTHMA-070 protein retains thefunctional activity of the FTHMA-070 protein of SEQ ID NO:54.

Accordingly, a useful T85 protein is a protein which includes an aminoacid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or99% identical to the amino acid sequence of SEQ ID NO:58 and retains thefunctional activity of the T85 protein of SEQ ID NO:58. In otherinstances, the T85 protein is a protein having an amino acid sequence55%, 65%, 75%, 85%, 95%, or 98% identical to one of the T85 fibronectintype III or Ig superfamily domains (SEQ ID NOs:61-67). In a preferredembodiment, the T85 protein retains the functional activity of the T85protein of SEQ ID NO:58.

Preferred Tango-77 protein has the amino acid sequence shown of SEQ IDNO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, or SEQ IDNO:83. Other useful Tango-77 proteins are substantially identical to SEQID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, or SEQID NO:83 and retain the functional activity of the protein of SEQ IDNO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, or SEQ IDNO:83 yet differ in amino acid sequence due to natural allelic variationor mutagenesis. Accordingly, a useful Tango-77 protein is a proteinwhich includes an amino acid sequence at least about 45%, preferably55%, 65%, 75%, 85%, 95%, or 99% identical to the amino acid sequence ofSEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, orSEQ ID NO:83 and retains the functional activity of the Tango-77proteins of SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQID NO:81, or SEQ ID NO:83. In a preferred embodiment, the Tango-77protein retains a functional activity of the Tango-77 protein of SEQ IDNO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, or SEQ IDNO:83.

In one embodiment, a biologically active portion of a SPOIL proteincomprises at least an IL-1 signature domain. In another embodiment, abiologically active portion of a SPOIL protein comprises a SPOILsignature motif. In yet another embodiment, a biologically activeportion of a SPOIL protein comprises a SPOIL unique domain. In yetanother embodiment, a biologically active portion of a SPOIL proteincomprises a SPOIL C-terminal unique domain. In another embodiment, abiologically active portion of a SPOIL protein comprises a signalsequence and/or is secreted. In another embodiment, a biologicallyactive portion of a SPOIL protein lacks a signal sequence and/or isintracellular.

It is to be understood that a preferred biologically active portion of aSPOIL protein of the present invention may contain at least one of theabove-identified structural domains. Another preferred biologicallyactive portion of a SPOIL protein may contain at least two of theabove-identified structural domains. Another preferred biologicallyactive portion of a SPOIL protein may contain at least three or more ofthe above-identified structural domains.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native SPOILprotein.

In a preferred embodiment, the SPOIL protein has an amino acid sequenceshown in SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113, theamino acid sequence encoded by the DNA insert of the plasmid depositedwith ATCC as Accession Number 98883, or the amino acid sequence encodedby the DNA insert of the plasmid deposited with ATCC as Accession Number98984. In other embodiments, the SPOIL protein is substantiallyhomologous to SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113,the amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, and retains the functional activity of theprotein of SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113,the amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, yet differs in amino acid sequence due tonatural allelic variation or mutagenesis, as described in detail insubsection I above. Accordingly, in another embodiment, the SPOILprotein is a protein which comprises an amino acid sequence at leastabout 60-65% identical to the amino acid sequence of SEQ ID NO:90, SEQID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encodedby the DNA insert of the plasmid deposited with ATCC as Accession Number98883, or the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98984, and, preferably,retains a functional activity of the SPOIL proteins of SEQ ID NO:90, SEQID NO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encodedby the DNA insert of the plasmid deposited with ATCC as Accession Number98883, or the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98984. Preferably, theprotein is at least about 70-75% identical to SEQ ID NO:90, SEQ IDNO:102, SEQ ID NO:105, SEQ ID NO:113, the amino acid sequence encoded bythe DNA insert of the plasmid deposited with ATCC as Accession Number98883, or the amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number 98984, more preferablyat least about 80-85% identical to SEQ ID NO:90, SEQ ID NO:102, SEQ IDNO:105, SEQ ID NO:113, the amino acid sequence encoded by the DNA insertof the plasmid deposited with ATCC as Accession Number 98883, or theamino acid sequence encoded by the DNA insert of the plasmid depositedwith ATCC as Accession Number 98984, even more preferably at least about90-95% identical to SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ IDNO:113, the amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984, and most preferably at least about 95% or moreidentical to SEQ ID NO:90, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:113,the amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number 98883, or the amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number 98984.

In one embodiment, a biologically active portion of a NEOKINE proteincomprises at least a NEOKINE CXC signature motif. In another embodiment,a biologically active portion of a NEOKINE protein comprises at least asignal sequence. In another embodiment, a biologically active portion ofa NEOKINE protein comprises a NEOKINE amino acid sequence lacking asignal sequence (e.g., a mature NEOKINE protein).

It is to be understood that a preferred biologically active portion of aNEOKINE protein of the present invention may contain at least one of theabove-identified structural domains. A more preferred biologicallyactive portion of a NEOKINE protein may contain at least two of theabove-identified structural domains. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native NEOKINE protein.

In a preferred embodiment, the NEOKINE protein has an amino acidsequence shown in SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ IDNO:125. In other embodiments, the NEOKINE protein is substantiallyhomologous to SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ IDNO:125, and retains the functional activity of the protein of SEQ IDNO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125, yet differs inamino acid sequence due to natural allelic variation or mutagenesis, asdescribed in detail in subsection I above. Accordingly, in anotherembodiment, the NEOKINE protein is a protein which comprises an aminoacid sequence at least about 60% homologous to the amino acid sequenceof SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125 andretains the functional activity of the NEOKINE proteins of SEQ IDNO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125, respectively.Preferably, the protein is at least about 70% homologous to SEQ IDNO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125, more preferablyat least about 80% homologous to SEQ ID NO:116, SEQ ID NO:119, SEQ IDNO:122, or SEQ ID NO:125, even more preferably at least about 90%homologous to SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ IDNO:125, and most preferably at least about 95% or more homologous to SEQID NO:116, SEQ ID NO:119, SEQ ID NO:122, or SEQ ID NO:125.

Preferred T129 protein has the amino acid sequence shown of SEQ IDNO:138. Other useful T129 proteins are substantially identical to SEQ IDNO:138 and retain the functional activity of the protein of SEQ IDNO:138 yet differ in amino acid sequence due to natural allelicvariation or mutagenesis. Accordingly, a useful T129 protein is aprotein which includes an amino acid sequence at least about 45%,preferably 55%, 65%, 75%, 85%, 95%, or 99% identical to the amino acidsequence of SEQ ID NO:138 and retains the functional activity of theT129 proteins of SEQ ID NO:138. In other instances, the T129 protein isa protein having an amino acid sequence 55%, 65%, 75%, 85%, 95%, or 98%identical to the T129 TNFR/NGFR cysteine rich domain (SEQ ID NO:141). Ina preferred embodiment, the T129 protein retains the functional activityof the T129 protein of SEQ ID NO:138.

Preferred polypeptides have the amino acid sequence of SEQ ID NO:147,148, 165, or 166. Other useful proteins are substantially identical(e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%)to any of SEQ ID NO:147, 148, 165, or 166, and retain the functionalactivity of the protein of the corresponding naturally-occurring proteinyet differ in amino acid sequence due to natural allelic variation ormutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence and non-homologous sequences can be disregardedfor comparison purposes). In one embodiment, an alignment is a globalalignment, e.g., an overall sequence alignment. In another embodiment,an alignment is a local alignment. In a preferred embodiment, the lengthof a sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least 70%, 80%, or90% of the length of the reference sequence to which it is aligned(e.g., when aligning a second sequence to the SPOIL amino acid sequenceof SEQ ID NO:90, at least 29, preferably at least 39, more preferably atleast 49, even more preferably at least 59, and even more preferably atleast 69, 78 or 88 amino acid residues are aligned). In a particularlypreferred embodiment, percent identity is calculated over the entirelength of a reference sequence. The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least 70%, 80%, or90% of the length of the reference sequence (e.g., when aligning asecond sequence to the NEOKINE amino acid sequence of SEQ ID NO:2 having99 amino acid residues, at least 30, preferably at least 40, morepreferably at least 50, even more preferably at least 59, and even morepreferably at least 69, 79, or 89 are aligned). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared.

When a position in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are homologous at that position (i.e., asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The percent homology between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100).

The determination of percent homology between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'lAcad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein molecules ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al., (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred, non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller, CABIOS (1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the CGC sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art and include ADVANCE and ADAM asdescribed in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5;and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 chimeric or fusion proteins. As used herein, aFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 “chimericprotein” or “fusion protein” comprises a FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 polypeptide operatively linked to anon-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259polypeptide. A FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 polypeptide refers to a polypeptide having an amino acid sequencecorresponding to FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259, whereas a non-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 polypeptide refers to a polypeptide having an aminoacid sequence corresponding to a protein which is not substantiallyidentical to the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 protein, e.g., a protein which is different from the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein and which isderived from the same or a different organism. Within a FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 fusion protein theFTHMA-070 or T85 polypeptide can correspond to all or a portion of aFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein,preferably at least one biologically active portion of a FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein. Within thefusion protein, the term “operatively linked” is intended to indicatethat the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259polypeptide and the non-FTHMA-070 or T85 polypeptide are fused in-frameto each other. The non-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 polypeptide can be fused to the N-terminus orC-terminus of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 polypeptide.

One useful fusion protein is a GST-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 fusion protein in which the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequences are fusedto the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259.

In another embodiment, the fusion protein is a FTHMA-070, Tango85,SPOIL, or NEOKINE protein containing a heterologous signal sequence atits N-terminus. For example, the native FTHMA-070, Tango85, SPOIL, orNEOKINE signal sequence can be removed and replaced with a signalsequence from another protein. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of FTHMA-070, Tango85, SPOIL,or NEOKINE can be increased through use of a heterologous signalsequence.

In another embodiment, the fusion protein is a Tango-77, Tango129 orA259 protein containing a heterologous signal sequence at itsN-terminus. For example, the native Tango-77, Tango129 or A259 signalsequence (i.e., about amino acids 1 to 63 of SEQ ID NO:72; SEQ ID NO:74;or about amino acids 1 to 52 of SEQ ID NO:77; SEQ ID NO:78; or aboutamino acids 1 to 21 of SEQ ID NO:81; SEQ ID NO:82) can be removed andreplaced with a signal sequence from another protein. For example, thenative T129 signal sequence (i.e., about amino acids 1 to 22 of SEQ IDNO:138) can be removed and replaced with a signal sequence from anotherprotein. In certain host cells (e.g., mammalian host cells), expressionand/or secretion of Tango-77 can be increased through use of aheterologous signal sequence. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Ausubel et al., supra). Other examples of eukaryoticheterologous signal sequences include the secretory sequences ofmelittin and human placental alkaline phosphatase (Stratagene; La Jolla,Calif.). In yet another example, useful prokaryotic heterologous signalsequences include the phoA secretory signal (Sambrook et al., supra) andthe protein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an FTHMA-070, Tango85,Tango77, Tango129 or A259-immunoglobulin fusion protein in which all orpart of FTHMA-070 or T85 is fused to sequences derived from a member ofthe immunoglobulin protein family. The FTHMA-070, Tango85, Tango77,Tango129 or A259-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a FTHMA-070, Tango85, Tango77,Tango129 or A259 ligand and a FTHMA-070, Tango85, Tango77, Tango129 orA259 protein. The FTHMA-070, Tango85, Tango77, Tango129 orA259-immunoglobulin fusion proteins can be used to affect thebioavailability of a FTHMA-070, Tango85, Tango77, Tango129 or A259cognate ligand. Inhibition of the FTHMA-070, Tango85, Tango77, Tango129or A259 ligand/FTHMA-070, Tango85, Tango77, Tango129 or A259 interactionmay be useful therapeutically. Moreover, the FTHMA-070, Tango85,Tango77, Tango129 or A259-immunoglobulin fusion proteins of theinvention can be used as immunogens to produce anti-FTHMA-070, Tango85,Tango77, Tango129 or A259 antibodies in a subject, to purify FTHMA-070,Tango85, Tango77, Tango129 or A259 ligands and in screening assays toidentify molecules which inhibit the interaction of FTHMA-070, Tango85,Tango77, Tango129 or A259 with a FTHMA-070, Tango85, Tango77, Tango129or A259 ligand.

In yet another embodiment, the fusion protein is a SPOIL orNEOKINE-immunoglobulin fusion protein in which the SPOIL or NEOKINEsequence are fused to sequences derived from a member of theimmunoglobulin protein family. Soluble derivatives have also been madeof cell surface glycoproteins in the immunoglobulin gene superfamilyconsisting of an extracellular domain of the cell surface glycoproteinfused to an immunoglobulin constant (Fc) region (see e.g., Capon et al.(1989) Nature 337:525-531 and Capon U.S. Pat. Nos. 5,116,964 and5,428,130 [CD4-IgG1 constructs]; Linsley, et al. (1991) J. Exp. Med.173:721-730 [a CD28-IgG1 construct and a B7-1-IgG1 construct]; andLinsley et al. (1991) J. Exp. Med. 174:561-569 and U.S. Pat. No.5,434,131[a CTLA4-IgG1]). Such fusion proteins have proven useful formodulating receptor-ligand interactions. Soluble derivatives of cellsurface proteins of the tumor necrosis factor receptor (TNFR)superfamily proteins have been made consisting of an extracellulardomain of the cell surface receptor fused to an immunoglobulin constant(Fc) region (See for example Moreland et al. (1997) N. Engl. J. Med.337(3):141-147; van der Poll et al. (1997) Blood 89(10):3727-3734; andAmmann et al. (1997) J. Clin. Invest. 99(7):1699-1703.).

In yet another embodiment, the fusion protein comprises NEOKINEsequences (e.g., the NEOKINE CXC signature motif) fused to sequencesform other CXC cytokines. For example, NEOKINE sequences C-terminal toand including the first conserved cysteine residues can be fused toN-terminal sequences of other non-NEOKINE chemokines (e.g., comprisingfrom the N-terminal amino acid residue to the amino acid residueN-terminal to the first conserved cysteine).

The SPOIL or NEOKINE-immunoglobulin fusion proteins of the invention canbe incorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a SPOIL or NEOKINE protein anda SPOIL or NEOKINE target molecule on the surface of a cell, to therebysuppress SPOIL or NEOKINE-mediated signal transduction in vivo. TheSPOIL or NEOKINE-immunoglobulin fusion proteins can be used to affectthe bioavailability of a SPOIL or NEOKINE cognate ligand. Inhibition ofthe SPOIL or NEOKINE ligand/SPOIL or NEOKINE interaction may be usefultherapeutically for both the treatment and modulation of inflammationand immune disorders, as well as modulating (e.g., promoting orinhibiting) immune cell responses, cell adhesion, and/or cell homing.Moreover, the SPOIL or NEOKINE-immunoglobulin fusion proteins of theinvention can be used as immunogens to produce anti-SPOIL or NEOKINEantibodies in a subject, to purify SPOIL or NEOKINE ligands and inscreening assays to identify molecules which inhibit the interaction ofSPOIL or NEOKINE with a SPOIL or NEOKINE target molecule.

Preferably, a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 chimeric or fusion protein of the invention is produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Current Protocols in Molecular Biology, Ausubel et al. eds., JohnWiley & Sons: 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).An FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein.

A signal sequence of a polypeptide of the invention (SEQ ID NO:149 and163) can be used to facilitate secretion and isolation of the secretedprotein or other proteins of interest. Signal sequences are typicallycharacterized by a core of hydrophobic amino acids which are generallycleaved from the mature protein during secretion in one or more cleavageevents. Such signal peptides contain processing sites that allowcleavage of the signal sequence from the mature proteins as they passthrough the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as to thesignal sequence itself and to the polypeptide in the absence of thesignal sequence (i.e., the cleavage products). In one embodiment, anucleic acid sequence encoding a signal sequence of the invention can beoperably linked in an expression vector to a protein of interest, suchas a protein which is ordinarily not secreted or is otherwise difficultto isolate. The signal sequence directs secretion of the protein, suchas from a eukaryotic host into which the expression vector istransformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by art recognized methods. Alternatively, the signal sequence canbe linked to the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

In another embodiment, the signal sequences of the present invention canbe used to identify regulatory sequences, e.g., promoters, enhancers,repressors. Since signal sequences are the most amino-terminal sequencesof a peptide, it is expected that the nucleic acids which flank thesignal sequence on its amino-terminal side will be regulatory sequenceswhich affect transcription. Thus, a nucleotide sequence which encodesall or a portion of a signal sequence can be used as a probe to identifyand isolate signal sequences and their flanking regions, and theseflanking regions can be studied to identify regulatory elements therein.

The present invention also pertains to variants of the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteins whichfunction as either FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 agonists (mimetics) or as FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antagonists. Variants of the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein can begenerated by mutagenesis, e.g., discrete point mutation or truncation ofthe FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein. An agonist of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein.An antagonist of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein can inhibit one or more of the activities ofthe naturally occurring form of the FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein by, for example, competitively bindingto a downstream or upstream member of a cellular signaling cascade whichincludes the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 protein. Thus, specific biological effects can be elicited bytreatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteins.

In one embodiment, a SPOIL protein which acts as an IL-1 receptorantagonist can be converted into an IL-1 agonist by site specificmutagenesis. For example, the aspartic acid at amino acid residue 91 ofSEQ ID NO:90 or amino acid residue 74 of SEQ ID NO:93, can besubstituted with a lysine to create an IL-1 agonist. In a similarmanner, the alanine at amino acid residue 162 of SEQ ID NO:102 or thealanine residue at amino acid residue 201 of SEQ ID NO:105 can besubstituted with a lysine to create an IL-1 agonist. Exemplary methodsof converting IL-1ra into an IL-1 agonist are set forth in Ju et al.(1991) Proc. Natl. Acad. Sci. USA 88:2658-2662.

Variants of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 protein which function as either FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 agonists (mimetics) or as FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein for FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein agonist or antagonist activity. In oneembodiment, a variegated library of FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 variants can be produced by, forexample, enzymatically ligating a mixture of synthetic oligonucleotidesinto gene sequences such that a degenerate set of potential FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequencestherein. There are a variety of methods which can be used to producelibraries of potential FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

In addition, libraries of fragments of the FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein coding sequence can be used togenerate a variegated population of FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 fragments for screening and subsequentselection of variants of a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein. In one embodiment, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 coding sequence with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 proteins. The most widely usedtechniques, which are amenable to high through-put analysis, forscreening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 variants (Arkin andYourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.(1993) Protein Engineering 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated SPOIL library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to aparticular ligand in a SPOIL-dependent manner. The transfected cells arethen contacted with the ligand and the effect of expression of themutant on signaling by the ligand can be detected, e.g., by measuringany of a number of immune cell responses. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of ligand induction, and the individual clones furthercharacterized.

In one embodiment, cell based assays can be exploited to analyze avariegated NEOKINE library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to aparticular ligand in a NEOKINE-dependent manner. The transfected cellsare then contacted with the ligand and the effect of expression of themutant on signaling by the ligand can be detected, e.g., by measuringany of a number of inflammatory or angiogenic responses. Plasmid DNA canthen be recovered from the cells which score for inhibition, oralternatively, potentiation of ligand induction, and the individualclones further characterized.

An isolated FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 protein, or a portion or fragment thereof, can be used as animmunogen to generate antibodies that bind FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 using standard techniques forpolyclonal and monoclonal antibody preparation. The full-lengthFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteincan be used or, alternatively, the invention provides antigenic peptidefragments of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 for use as immunogens.

The antigenic peptide of FTHMA-070 or T85 comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence shown in SEQ ID NO:54 and encompasses an epitope of FTHMA-070or T85 such that an antibody raised against the peptide forms a specificimmune complex with FTHMA-070 or T85. Preferred epitopes encompassed bythe antigenic peptide are regions of FTHMA-070 or T85 that are locatedon the surface of the protein, e.g., hydrophilic regions.

The antigenic peptide of Tango-77 comprises at least 8 (preferably 10,15, 20, or 30) amino acid residues of the amino acid sequence shown inSEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81 orSEQ ID NO:83 and encompasses an epitope of Tango-77 such that anantibody raised against the peptide forms a specific immune complex withTango-77.

The antigenic peptide of SPOIL comprises at least 8 amino acid residuesof the amino acid sequence shown in SEQ ID NO:90, SEQ ID NO:102, SEQ IDNO:105, SEQ ID NO:113, the amino acid sequence encoded by the DNA insertof the plasmid deposited with ATCC as Accession Number 98883, or theamino acid sequence encoded by the DNA insert of the plasmid depositedwith ATCC as Accession Number 98984, and encompasses an epitope of SPOILsuch that an antibody raised against the peptide forms a specific immunecomplex with SPOIL. Preferably, the antigenic peptide comprises at least10 amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

The antigenic peptide of NEOKINE comprises at least 8 amino acidresidues of the amino acid sequence shown in SEQ ID NO:116, SEQ IDNO:119, SEQ ID NO:122, or SEQ ID NO:125 and encompasses an epitope ofNEOKINE such that an antibody raised against the peptide forms aspecific immune complex with NEOKINE. Preferably, the antigenic peptidecomprises at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues. Preferredepitopes encompassed by the antigenic peptide are regions of NEOKINEthat are located on the surface of the protein, e.g., hydrophilicregions.

The antigenic peptide of T129 comprises at least 8 (preferably 10, 15,20, or 30) amino acid residues of the amino acid sequence shown in SEQID NO:138 and encompasses an epitope of T129 such that an antibodyraised against the peptide forms a specific immune complex with T129.

The antigenic peptide of a259 comprises at least 8 (preferably 10, 15,20, or 30) amino acid residues of the amino acid sequence of SEQ IDNO:147 or 165, and encompasses an epitope of the protein such that anantibody raised against the peptide forms a specific immune complex withthe protein.

Preferred epitopes encompassed by the antigenic peptide are regions ofT129 that are located on the surface of the protein, e.g., hydrophilicregions. A hydrophobicity analysis of the human T129 protein sequenceindicates that the regions between, e.g., amino acids 120 and 130,between amino acids 140 and 160, and between amino acids 400 and 420 ofSEQ ID NO:2 are particularly hydrophilic and, therefore, are likely toencode surface residues useful for targeting antibody production.

A T129 immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed T129 protein or a chemicallysynthesized T129 polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic T129 preparation induces a polyclonal anti-T129 antibodyresponse.

Preferred epitopes encompassed by the antigenic peptide are regions thatare located on the surface of the A259 protein, e.g., hydrophilicregions. FIGS. 10 and 11 are hydropathy plots of the proteins of theinvention. These plots or similar analyses can be used to identifyhydrophilic regions.

A FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein or a chemically synthesized FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent. Immunizationof a suitable subject with an immunogenic FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 preparation induces a polyclonalanti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259antibody response.

Accordingly, another aspect of the invention pertains to anti-FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 antibodies. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically bindsan antigen, such as FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259. A molecule which specifically binds to FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 is a molecule whichbinds FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259, butdoes not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259. Examples of immunologically activeportions of immunoglobulin molecules include F(ab) and F(ab′)₂ fragmentswhich can be generated by treating the antibody with an enzyme such aspepsin. The invention provides polyclonal and monoclonal antibodies thatbind FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 protein with which itimmunoreacts.

Polyclonal anti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or

A259 antibodies can be prepared as described above by immunizing asuitable subject with a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 immunogen. The anti-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259. If desired, the antibodymolecules directed against FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 can be isolated from the mammal (e.g., from the blood)and further purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing variousantibodies monoclonal antibody hybridomas is well known (see generallyCurrent Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley& Sons, Inc., New York, N.Y.). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 immunogen as described above, and the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridomaproducing a monoclonal antibody that binds FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259monoclonal antibody (see, e.g., Current Protocols in Immunology, supra;Galfre et al. (1977) Nature 266:55052; R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol. Med.,54:387-402. Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line, e.g., a myeloma cell line that is sensitive to culturemedium containing hypoxanthine, aminopterin and thymidine (“HATmedium”). Any of a number of myeloma cell lines can be used as a fusionpartner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines areavailable from ATCC. Typically, HAT-sensitive mouse myeloma cells arefused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridomacells resulting from the fusion are then selected using HAT medium,which kills unfused and unproductively fused myeloma cells (unfusedsplenocytes die after several days because they are not transformed).Hybridoma cells producing a monoclonal antibody of the invention aredetected by screening the hybridoma culture supernatants for antibodiesthat bind FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259,e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 antibody can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 to thereby isolate immunoglobulin library members that bindFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SudZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

Additionally, recombinant anti-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Application No.PCT/US86/02269; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, (1985) Science 229:1202-1207; Oi et al. (1986)Bio/techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheave and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion ofTango-77 or A259. Monoclonal antibodies directed against the antigen canbe obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to the described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope.

First, a non-human monoclonal antibody which binds a selected antigen(epitope), e.g., an antibody which inhibits Tango-77 or A259 activity,is identified. The heave chain and the light chain of the non-humanantibody are cloned and used to create phage display Fab fragments. Forexample, the heave chain gene can be cloned into a plasmid vector sothat the heavy chain can be secreted from bacteria. The light chain genecan be cloned into a phage coat protein gene so that the light chain canbe expressed on the surface of phage. A repertoire (random collection)of human light chains fused to phage is used to infect the bacteriawhich express the non-human heavy chain. The resulting progeny phagedisplay hybrid antibodies (human light chain/non-human heavy chain). Theselected antigen is used in a panning screen to select phage which bindthe selected antigen. Several rounds of selection may be required toidentify such phage. Next, human light chain genes are isolated from theselected phage which bind the selected antigen. These selected humanlight chain genes are then used to guide the selection of human heavychain genes as follows. The selected human light chain genes areinserted into vectors for expression by bacteria. Bacteria expressingthe selected human light chains are infected with a repertoire of humanheavy chains fused to phage. The resulting progeny phage display humanantibodies (human light chain/human heavy chain).

Next, the selected antigen is used in a panning screen to select phagewhich bind the selected antigen. The phage selected in this step displaycompletely human antibody which recognize the same epitope recognized bythe original selected, non-human monoclonal antibody. The genes encodingboth the heavy and light chains are readily isolated and be furthermanipulated for production of human antibody. This technology isdescribed by Jespers et al. (1994, Bio/technology 12:899-903).

An anti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259antibody (e.g.; monoclonal antibody) can be used to isolate FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 by standardtechniques, such as affinity chromatography or immunoprecipitation. Ananti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259antibody can facilitate the purification of natural FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 from cells and ofrecombinantly produced FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 expressed in host cells. Moreover, an anti-FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 antibody can be usedto detect FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein.Anti-FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259antibodies can be used diagnostically to monitor protein levels intissue as part of a clinical testing procedure, e.g., to, for example,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Delta3 Methods of Treating Disease

Based at least in part on the fact that the Notch signaling pathway hasbeen implicated in development of the nervous system, in particular inregulating neuronal differentiation and vasculature, e.g., CNSvasculature, a wide variety of pathological diseases or conditions canbenefit from treatment with Delta3 nucleic acids, proteins, andmodulators thereof. In particular, based at least in part on theobservation that PS1 and PS2, genes encoding amyloid precursor proteins,which are mutated in about 10% of cases of Alzheimer's disease, arefunctionally linked to the Notch signaling pathway, mutations in genesof the Notch signaling pathway, e.g., Delta genes, could also result inAlzheimer's disease or other neurodegenerative or neuro-developmentaldiseases. The Notch signaling pathway plays a role in the development ofvasculature. For example, loss of Dll1 function mutants become severallyhemorrhagic after embryonic day 10. Furthermore, mutations in Notch3result in CADASIL, a disease characterized by stroke. In addition, micewith a functionally ablated PS1 gene exhibit hemorrhages in the brainand/or spinal cord after embryonic day 11.5 (Wong et al. (1997) Nature387:288). In addition, the Notch pathway has been implicated inhematologic development. Specifically, molecules in the Notch signalingpathway have been shown to be expressed in a wide variety of bloodcells, including but not limited to those of myeloid and lymphoidorigins. Notch-1 was shown to play a role in T-cell development.Furthermore, since the Notch signaling pathway is involved in cell fatedetermination at least in the nervous system, immune system andendothelial system, it is likely that the Notch signaling pathway, andin particular Delta3 is involved in cell fate determination inadditional biological systems. Accordingly, the invention also providesmethods for treating diseases or disorders arising from an abnormal cellproliferation and/or differentiation of cells other than cells from thenervous system, immune system, and vasculature.

Among the disorders that can be treated or prevented according to themethods of the invention include pathological neurogenic, neoplastic orhyperplastic conditions. Neurologic diseases, e.g., neurodegenerative,neuro-differentiative and neuro-developmental diseases, that mightbenefit from this methodology include, but are not limited toneuropathies, e.g., peripheral neuropathy such as ACCPN, stroke,dementia, e.g., cerebral autosomal dominant arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL), degenerativelesions (Parkinson's disease, Alzheimer's disease, Huntington's chorea,amyotrophic lateral sclerosis, spinocerebellar degenerations),demyelating diseases (multiple sclerosis, human immunodeficiencyassociated myelopathy, transverse myelopathy, progressive multifocalleukoencephalopathy, pontine myelinolysis), motor neuron injuries,progressive spinal muscular atrophy, progressive bulbar palsy, primarylateral sclerosis, infantile and juvenile muscular atrophy, progressivebulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis, andhereditary motorsensory neuropathy (Charcot-Marie-Tooth disease), spinalcord injuries, brain injuries, lesions associated with surgery, ischemiclesions, malignant lesions, infectious lesions.

Additional neurological diseases that can be treated according to themethod of the invention include neuropathies, e.g., peripheralneuropathies, e.g., Agenesis of the Corpus Callosum with PeripheralNeuropathy (ACCPN). In fact, as set forth in the examples presentedbelow, hDelta3 has been mapped to human chromosome 15 close to frameworkmarkers D15S1244 and D15S144, a chromosomal region which has been shownto be genetically linked (ACCPN) (Casaubon et al. (1996) Am J. Hum.Genet. 58:28). The disease is characterized by a progressive peripheralneuropathy caused by axonal degeneration and a central nervous system(CNS) malformation characterized by the absence of hypoplasia of thecorpus callosum. The disorder appears early in life, is progressive andresults in death in the third decade of life of the subject.

Neuropathies refer to disorders of peripheral nerves and includes bothmotor and sensory functions, since most motor and sensory axons run inthe same nerves. Neurophathies may be either chronic or acute. Oneexample of a acute neuropathy is the Guillain-Barre syndrome, whichoften follow respiratory infection. Chronic neuropathies include, e.g.,acute intermittent porphyria, Charcot-Marie-Tooth disease, metabolicdiseases such as diabetes, obesity, and B12 deficiency, intoxication,nutritional disorders.

Disorders of the vasculature, also termed “vascular disorders”, inaddition to CADASIL and stroke, that can be treated or preventedaccording to the methods of the invention include atheroma, tumorangiogenesis, wound healing, diabetic retinopathy, hemangioma,psoriasis, and restenosis, e.g., restenosis resulting from balloonangioplasty.

In one embodiment, diseases or disorders caused or contributed to byaberrant Delta3 activity, such as aberrant Delta3 protein levels or anaberrant biological activity or which are associated with one or morespecific Delta3 alleles, e.g., a mutant Delta3 allele, can be treatedwith Delta3 therapeutics. Aberrant protein levels can be caused, e.g.,by aberrant gene expression. Such aberrant activity can result, forexample, in aberrant cell proliferation and/or differentiation or celldeath. For example, aberrant Delta3 activity in a subject can result inincreased proliferation of certain cells in the subject. Subjects havinga disorder characterized by abnormal cell proliferation can be treatedby administration of a Delta3 therapeutic inhibiting or decreasing suchproliferation. The specific Delta3 therapeutic used may vary dependingon the type of the cell that is proliferating aberrantly. Theappropriate Delta3 therapeutic to use can be determined, e.g., by invitro culture of a sample of such cells which can be obtained from thesubject, in the presence and in the absence of Delta3 therapeutics.

Diseases or conditions associated with aberrant cell proliferation whichcan be treated or prevented with Delta3 therapeutics include cancers,malignant conditions, premalignant conditions, benign conditions. Thecondition to be treated or prevented can be a solid tumor, such as atumor arising in an epithelial tissue. For example, the cancer can becolon or cervix cancer. Cancer of the colon and cervix have in fact beenfound to have increased levels of expression of Notch as compared tonormal tissue (PCT Publication No. WO/07474, Apr. 14, 1994).Accordingly, treatment of such a cancer could comprise administration tothe subject of a Delta3 therapeutic decreasing the interaction of Notchwith Delta3. Other cancers that can be treated or prevented with aDelta3 protein include sarcomas and carcinomas, e.g., lung cancer,cancer of the esophagus, lung cancer, melanoma, seminoma, and squamousadenocarcinoma. Additional solid tumors within the scope of theinvention include those that can be found in a medical textbook. Thecondition to be treated or prevented can also be a soluble tumor, suchas leukemia, either chronic or acute, including chronic or acutemyelogenous leukemia, chronic or acute lymphocytic leukemia,promyelocytic leukemia, monocytic leukemia, myelomonocytic leukemia, anderythroleukemia. Yet other proliferative disorders that can be treatedwith a Delta3 therapeutic of the invention include heavy chain disease,multiple myeloma, lymphoma, e.g., Hodgkin's lymphoma and non-Hodgkin'slymphoma, Waldenstroem's macroglobulemia, and fibroproliferativedisorders, particularly of cerebravascular tissue.

Diseases or conditions characterized by a solid or soluble tumor can betreated by administrating a Delta3 therapeutic either locally orsystemically, such that proliferation of the cells having an aberrantproliferation is inhibited or decreased. Methods for administering thecompounds of the invention are further described below.

The invention also provides methods for preventing the formation and/ordevelopment of tumors. For example, the development of a tumor can bepreceded by the presence of a specific lesion, such as a pre-neoplasticlesion, e.g., hyperplasia, metaplasia, and dysplasia. Such lesions canbe found, e.g., in epithelial tissue. Thus, the invention provides amethod for inhibiting progression of such a lesion into a neoplasticlesion, comprising administering to the subject having a preneoplasticlesion a amount of a Delta3 therapeutic sufficient to inhibitprogression of the preneoplastic lesion into a neoplastic lesion.

In a preferred embodiment, the invention provides a method forinhibiting endothelial cell proliferation and/or differentiation,comprising contacting a Delta3 therapeutic with a tissue in whichendothelial cells are proliferating, such as a developing tumor or ahyperproliferative disease, i.e., a disease associated with abnormalcellular proliferation. Blocking the proliferation of endothelial cellswill result in inhibition of development of endothelium and bloodvessels, thus limiting access to the tumor of compounds necessary fortumor development.

The invention also provides for methods for treating or preventingdiseases or conditions associated with insufficient cell proliferation.For example, Delta3 therapeutics can be used to stimulate tissue repair,regeneration, and/or wound healing, e.g., of neural tissue, such asafter surgery or to stimulate tissue healing from burns. Other diseasein which proliferation of cells is desired are hypoproliferativediseases, i.e., diseases characterized by an abnormally lowproliferation of certain cells.

In yet another embodiment, the invention provides a method for treatingor preventing diseases or conditions characterized by aberrant celldifferentiation. Accordingly, the invention provides methods forstimulating cellular differentiation in conditions characterized by aninhibition of normal cell differentiation which may or may not beaccompanied by excessive proliferation. Alternatively, Delta3therapeutics can be used to inhibit differentiation of specific cells.

In one method, the aberrantly proliferating and/or differentiating cellis a cell present in the nervous system. Accordingly, the inventionprovides methods for treating diseases or conditions associated with acentral or peripheral nervous system. For example, the inventionprovides methods for treating lesions of the nervous system involving anaberrant Delta3 activity in neurons, in Schwann cells, glial cells, orother types of neural cells. Disorders of the nervous system are setforth above.

In another embodiment, a Delta3 therapeutic can be utilized toameliorate a symptom of obesity and/or disorders that accompany or areexacerbated by an obese state, such as cardiovascular and circulatorydisorders, metabolic abnormalities typical of obesity, such ashyperinsulinemia, insulin resistance, diabetes, including non-insulindependent diabetes mellitus (NIDDM), insulin dependent diabetes mellitus(IDDM), and maturity onset diabetes of the young (MODY), disorders ofenergy homeostasis, disorders associated with lipid metabolism, such ascachexia.

With respect to cardiovascular disorders, symptoms of coronary diseases(e.g., cardiovascular diseases including unstable angina pectoris,myocardial infarction, acute myocardial infarction, coronary arterydisease, coronary revascularization, coronary restenosis, ventricularthromboembolism, atherosclerosis, coronary artery disease (e.g.,arterial occlusive disorders), plaque formation, cardiac ischemia,including complications related to coronary procedures, such aspercutaneous coronary artery angioplasty (balloon angioplasty)procedures) can be ameliorated. With respect to coronary procedures,such modulation can be achieved via administration of Delta3therapeutics prior to, during, or subsequent to the procedure.

Delta3 therapeutics (e.g., nucleic acids, proteins and modulatorsthereof) can, therefore, be used to modulate disorders resulting fromany blood vessel insult that can result in platelet aggregation. Suchblood vessel insults include, but are not limited to, vessel wallinjury, such as vessel injuries that result in a highly thrombogenicsurface exposed within an otherwise intact blood vessel e.g., vesselwall injuries that result in release of ADP, thrombin and/orepinephrine, fluid shear stress that occurs at the site of vesselnarrowing, ruptures and/or tears at the sites of atheroscleroticplaques, and injury resulting from balloon angioplasty or atherectomy.Preferably, such therapeutics do not effect initial platelet adhesion tovessel surfaces, or effect such adhesion to a relatively lesser extentthan the effect on platelet-platelet aggregation, e.g., unregulatedplatelet-platelet aggregation, following the initial platelet adhesion.

In addition, Delta3 therapeutics can be utilized to amieliorate asymptom of disorders associated with abnormal vasculogenesis (e.g.,cancers, including, but not limited to, cancers of the epithelia (e.g.,carcinomas of the pancreas, stomach, liver, secretory glands (e.g.,adenocarcinoma), bladder, lung, breast, skin (e.g., fibromatosis ormalignant melanoma), reproductive tract including prostate gland, ovary,cervix and uterus); cancers of the hematopoietic and immune system(e.g., leukemias and lymphomas); cancers of the central nervous, brainsystem and eye (e.g., gliomas, neuroblastoma and retinoblastoma); andcancers of connective tissues, bone, muscles and vasculature (e.g.,hemangiomas and sarcomas)), disorders related to fetal development, inparticular, disorders involving development of lung and kidney,lung-related disorders, and immune-related disorders, such asinflammatory-related disorders, e.g., asthma, allergy, and autoimmunedisorders, as well as neurological disorders, including developmental,cognitive and personality-related disorders, renal disorders, adrenalgland-related disorders; and disorders relating to skeletal muscle, suchas dystrophic disorders.

With respect to immune disorders, Delta3 therapeutics (e.g., nucleicacids, proteins and modulators thereof) can be utilized to modulateprocesses involved in lymphocyte development, differentiation andactivity, including, but not limited to development, differentiation andactivation of T cells, including T helper, T cytotoxic and non-specificT killer cell types and subtypes, and B cells, immune functionsassociated with such cells, and amelioration of one or more symptomsassociated with abnormal function of such cell types. Such disorders caninclude, but are not limited to, autoimmune disorders, such as organspecific autoimmune disorders, e.g., autoimmune thyroiditis, Type Idiabetes mellitus, insulin-resistant diabetes, autoimmune anemia,multiple sclerosis, and/or systemic autoimmune disorders, e.g.,rheumatoid arthritis, lupus or sclerodoma, allergy, including allergicrhinitis and food allergies, asthma, psoriasis, graft rejection,transplantation rejection, graft versus host disease, pathogenicsusceptibilities, e.g., susceptibility to certain bacterial or viralpathogens, wound healing and inflammatory reactions.

With respect to skeletal muscle-related disorders, Delta3 therapeuticscan be utilized to ameliorate symptoms of disorders including, forexample, muscular dystrophy disorders, e.g., Duchenne's musculardystrophy and X-linked recessive Emery-Dreifuss dystrophy (EDMD), aswell as developmental and other disorders that involve skeletal musclesuch as, for example, oculofacial-skeletal myorhythmias, sarcoidosis,and malignant hyperthermia susceptibility (MHS).

With respect to lung disorders, Delta3 therapeutics can be utilized, forexampe, to ameliorate a symptom of such pulmonary disorders, such asatelectasis, pulmonary congestion or edema, chronic obstructive airwaydisease (e.g., emphysema, chronic bronchitis, bronchial asthma, andbronchiectasis), diffuse interstitial diseases (e.g., sarcoidosis,pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome,idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis,desquamative interstitial pneumonitis, chronic interstitial pneumonia,fibrosing alveolitis, hamman-rich syndrome, pulmonary eosinophilia,diffuse interstitial fibrosis, Wegener's granulomatosis, lymphomatoidgranulomatosis, and lipid pneumonia), or tumors (e.g., bronchogeniccarcinoma, bronchiolovlveolar carcinoma, bronchial carcinoid, hamartoma,and mesenchymal tumors).

In another embodiment, the invention provides a method for enhancing thesurvival and/or stimulating proliferation and/or differentiation ofcells and tissues in vitro. For example, tissues from a subject can beobtained and grown in vitro in the presence of a Delta3 therapeutic,such that the tissue cells are stimulated to proliferate and/ordifferentiate. The tissue can then be readministered to the subject.

In another embodiment, as Notch can function to maintain cells in animmature state in vitro, i.e., as stem cells, e.g., the inventionprovides a method to expand the pool of hematopoietic stem cells throughthe interaction of Delta3 with Notch, which can be utilized in caseswhere it is desirable to do so including, but not limited to preparingcells harvested for subsequent bone marrow transplantation. Thus, Delta3 can be utilized in stem cell preservation, that is, can be utilized topreserve stem cells in an immature, undifferentiated state, and/orpreserving the stem cells' pluripotency, differentiation potential andproliferation potential. In one embodiment of such a stem cellpreservation method, stem cells are contacted with cells expressingDelta3 and exhibiting Delta3 on their cell surfaces. SuchDelta3-expressing cells can be presented, e.g., as stromal cells inculture. In another embodiment, stem cells are contacted withfull-length or soluble Delta3 attached to a solid surface, e.g., aculture plate, or microbeads.

In another embodiment, techniques such as that described above for stemcell preservation can be utilized to prevent death of CD4⁺/CD8⁺ T cells.Thus, such techniques can be used to repopulate peripheral T cellpopulations (e.g., as part of a leukemia therapy), or, andalternatively, can be used to produce and screen for an antigen-specificT cell clone.

In another embodiment, the invention can function as a method todetermine the fate of T cells in the developing thymus. Antagonists orantagonists of hDelta3 activity can determine whether a T cell willdevelop the CD4 or CD8 phenotype and thus be useful as a therapeuticagent in immunodeficiency disorders, such as, but not limited to AIDS.

In another embodiment, as the Notch signaling pathway has been shown tobe involved in eye development in Drosophila, and given the fact thatmDelta3 was highly expressed in the developing mouse eye, Delta3 nucleicacids, proteins, and modulators thereof and/or agonists and antagonistscan be used as therapeutics in such eye disorders as diabeticretinopathy, characterized by a hyper-proliferation of capillaries inthe retina.

Since, in some cases, genes may be upregulated in a disease state and inother cases may be down-regulated, it will be desirable to activateand/or potentiate or suppress and/or down-modulate Delta3 activitydepending on the condition to be treated using the techniques compoundsand methods described herein. Some genes may be under-expressed incertain disease states. The activity of Delta3 nucleic acids, proteins,and modulators thereof may be in some way impaired, leading to thedevelopment of neurodegenerative disease symptoms. Such down-regulationof Delta3 gene expression or decrease in the activity of a Delta3protein may have a causative or exacerbating effect on the diseasestate.

Among the approaches which may be used to ameliorate disease symptomsinvolving the misexpression of a Delta3 gene are, for example,antisense, ribozyme, and triple helix molecules described above.Compounds that compete with Delta3 nucleic acids, proteins, andmodulators thereof for binding to upstream or downstream elements in aDelta/Notch signaling cascade will antagonize Delta3 nucleic acids,proteins, and modulators thereof, thereby inducing a therapeutic effect.Examples of suitable compounds include the antagonists or homologsdescribed in detail above. In other instances, the increased expressionor activity of Delta3 nucleic acids, proteins, and modulators thereofmay be desirable and may be accomplished by, for example the use ofDelta3 agonists or mimetics or by gene replacement therapy, as describedherein.

Yet other Delta3 therapeutics comprise of a first peptide comprising aDelta3 peptide capable of binding to a receptor, e.g., a Notch receptor,and a second peptide which is cytotoxic. Such therapeutics can be usedto specifically target and lyse cells expressing or over-expressing areceptor for Delta3. For example, a fusion protein containing a Delta3peptide fused to a cytotoxic peptide can be used to eliminate or reducea tumor over-expressing Notch, e.g., colon and cervix neoplastic tumors.Alternatively, cells expressing or over-expressing Delta3 can betargeted for lysis, by, for example, targeting to the cell an antibodybinding specifically to a Delta3 protein linked to a cytotoxic peptide.

Based at least in part on the similarity of protein structure, it islikely that Delta3 nucleic acids, proteins, and modulators thereof canalso be used to treat diseases or conditions caused by or contributed byan aberrant activity of a Delta family gene product, e.g., an aberrantDelta1 or Delta2 activity or diseases or disorders which are associatedwith one or more specific Delta alleles, e.g., Delta1 or Delta2 alleles.Such diseases or conditions could include neurological diseases andcancer. Similarly, Delta therapeutics, e.g., Delta1 or Delta2therapeutics, could be used to prevent or treat diseases or disorderscaused by or contributed to by an aberrant Delta3 activity, or diseasesor disorders which are associated with a specific Delta3 allele. Deltatherapeutics can be prepared using, e.g., the nucleotide and proteinsequence information disclosed in the PCT Patent Publication WO 97/01571and tested using the assays described herein for testing Delta3therapeutics.

Compounds identified as increasing or decreasing Delta3 gene expressionor protein activity can be administered to a subject at therapeuticallyeffective dose to treat or ameliorate cardiovascular disease. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms associated with theparticular disease.

Pharmaceutical Compositions

The Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 nucleic acid molecules, Delta3, FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 proteins, and anti-Delta3, FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 antibodies (also referred toherein as “active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more additionalactive compounds.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, intramuscular and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). Inall'cases, the composition must be sterile and should be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a Delta3, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein or anti-Delta3, FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

With regard to antibodies which are to act in the brain, a dosage of 50mg/kg to 100 mg/kg is typically appropriate. Generally, partially humanantibodies and fully human antibodies have a longer half-life within thehuman body than other antibodies. Accordingly, lower dosages and lessfrequent administration is often possible. Modifications such aslipidation can be used to stabilize antibodies and to enhance uptake andtissue penetration (e.g., into the brain). A method for lipidation ofantibodies is described by Cruikshank et al. ((1997) J. Acquired ImmuneDeficiency Syndromes and Human Retrovirology 14:193).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Inone embodiment, a subject is treated with a protein, or polypeptide inthe range of between about 0.1 to 20 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between about 2 to 8weeks, more preferably between about 3 to 8 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

In clinical settings, the gene delivery systems for the therapeuticDelta3 gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g., by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.(1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). A Delta3 gene, such asany one of the sequences represented in the group consisting of SEQ IDNOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, or asequence homologous thereto can be delivered in a gene therapy constructby electroporation using techniques described, for example, by Dev etal. ((1994) Cancer Treat. Rev. 20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology), c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express FTHMA-070, Tango85, Tango77 or A259 protein (e.g.,via a recombinant expression vector in a host cell in gene therapyapplications), to detect FTHMA-070, Tango85, Tango77 or A259 mRNA (e.g.,in a biological sample) or a genetic lesion in a FTHMA-070, Tango85,Tango77 or A259 gene, and to modulate FTHMA-070, Tango85, Tango77 orA259 activity. In addition, the FTHMA-070, Tango85, Tango77 or A259proteins can be used to screen drugs or compounds which modulate theFTHMA-070, Tango85, Tango77 or A259 activity or expression as well as totreat disorders characterized by insufficient or excessive production ofFTHMA-070, Tango85, Tango77 or A259 protein or production of FTHMA-070,Tango85, Tango77 or A259 protein forms which have decreased or aberrantactivity compared to FTHMA-070, Tango85, Tango77 or A259 wild typeprotein. In addition, the anti-FTHMA-070, Tango85, Tango77 or A259antibodies of the invention can be used to detect and isolate FTHMA-070,Tango85, Tango77 or A259 proteins and modulate FTHMA-070, Tango85,Tango77 or A259 activity.

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology), c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials); and d) methodsof treatment (e.g., therapeutic and prophylactic methods as well as suchmethods in the context of pharmacogenomics). As described herein, aSPOIL protein of the invention has one or more of the followingactivities: (i) interaction of a SPOIL protein in the extracellularmilieu with a protein molecule on the surface of the same cell whichsecreted the SPOIL protein molecule (e.g., a SPOIL receptor or IL-1receptor); (ii) interaction of a SPOIL protein in the extracellularmilieu with a protein molecule on the surface of a different cell fromthat which secreted the SPOIL protein molecule (e.g., a SPOIL receptoror IL-1 receptor); (iii) complex formation between a SPOIL protein and acell-surface receptor; (iv) interaction of a SPOIL protein with a targetmolecule in the extracellular milieu, and (v) interaction of the SPOILprotein with a target molecule in the cytoplasm of a cell, and can thusbe used in, for example (1) regulating a signal transduction pathway(e.g., an IL-1-dependent or SPOIL-dependent pathway; (2) modulatingcytokine production and/or secretion; (3) modulating lymphokineproduction and/or secretion; (4) modulating production of adhesionmolecules; (5) modulation of nuclear transcription factors; (6)modulating secretion of IL-1; (7) competing with IL-1 to bind an IL-1receptor; (8) modulating a proinflammatory cytokine; (9) modulating cellproliferation, development or differentiation (e.g., IL-1-stimulated orSPOIL stimulated); (10) modulating bone metabolism (e.g., bone formationand reabsorption); and (11) mediating cellular “acute phase” response.The isolated nucleic acid molecules of the invention can be used, forexample, to express SPOIL protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect SPOILmRNA (e.g., in a biological sample) or a genetic alteration in an SPOILgene, and to modulate IL-1 activity, as described further below. Inaddition, the SPOIL proteins can be screened which modulate the SPOILactivity as well as to treat disorders characterized by insufficient orexcessive production of IL-1 which have decreased or aberrant activitycompared to normal IL-1 expression (e.g., inflammatory diseases, e.g.,rheumatoid arthritis, sepsis, stroke or diabetes, or stimulateddifferentiative or developmental disorders such as bone metabolismdisorders, e.g., osteoporosis, Paget's disease of bone, hypercalcemia ofmalignancy or osteolytic metastases, or diseases that involve the boweland are characterized by the production of inflammation and/orulceration in the small or large bowel including, e.g., inflammatorybowl disease (IBD), Crohn's disease, irritable bowel syndrome (IBS) andulcerative colitis. Soluble forms of the SPOIL protein can be used tobind IL-1 receptors and influence bioavailability of such a receptorscognate ligand. In addition, the anti-SPOIL antibodies of the inventioncan be used to detect and isolate SPOIL proteins.

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a NEOKINE protein of the invention has one or more ofthe following activities: (i) interaction of a NEOKINE protein with amembrane-bound NEOKINE receptor; (ii) interaction of a NEOKINE proteinwith a membrane-bound chemokine receptor; (iii) indirect interaction ofa NEOKINE protein with an intracellular protein via a membrane-boundNEOKINE receptor; (iv) indirect interaction of a NEOKINE protein with anintracellular protein via a membrane-bound chemokine receptor; (v)complex formation between a soluble NEOKINE protein and a NEOKINEbinding partner; (vi) inhibition of the interaction of chemokines (e.g.,pro-inflammatory chemokines) by binding to their cognate receptors;(vii) inhibition of the binding of HIV to HIV co-receptors; and (vii)inhibition of the binding of HIV to HIV co-receptors wherein saidbinding induces subsequent infection of susceptible cells and can thusbe used in, for example, (1) modulation of cellular signal transduction,either in vitro or in vivo; (2) regulation of gene transcription in acell expressing a NEOKINE receptor or a chemokine receptor; (3)regulation of gene transcription in a cell expressing a NEOKINE receptoror a chemokine receptor, wherein said cell is involved in angiogenesisor inflammation; (4) regulation of angiogenesis; (5) regulation ofangiogenesis, wherein said regulation comprises inhibibition ofangiogenesis; (6) regulation of angiogenesis, wherein said regulationcomprises maintenance of angiostasis; (7) regulation of inflammation;and (8) regulation of inflammation, wherein said regulation comprisesinhibition of chemoattraction (e.g., neutrophil chemoattraction),inhibition of inflammation, inhibition of inflammation by blocking theaction of pro-inflammatory chemokines by binding to their cognatereceptors, inhibition of psoriasis, suppression of immune rejectionfollowing skin graft, suppression of immune rejection following kidneytransplant, inhibition of kidney inflammation in acute renal failure,inhibition of brain inflammation following stroke or ischaemia, orinhibition of brain inflammation following viral infection. The isolatednucleic acid molecules of the invention can be used, for example, toexpress NEOKINE protein (e.g., via a recombinant expression vector in ahost cell in gene therapy applications), to detect NEOKINE mRNA (e.g.,in a biological sample) or a genetic alteration in a NEOKINE gene, andto modulate NEOKINE activity, as described further below. The NEOKINEproteins can be used to treat disorders characterized by insufficient orexcessive production of a non-NEOKINE chemokine or production ofchemokine forms which have decreased or aberrant activity compared towild type chemokines. In addition, the NEOKINE proteins can be used toscreen drugs or compounds which modulate the NEOKINE activity as well asto treat disorders characterized by insufficient or excessive productionof NEOKINE protein or production of NEOKINE protein forms which havedecreased or aberrant activity compared to NEOKINE wild type protein.Moreover, the anti-NEOKINE antibodies of the invention can be used todetect and isolate NEOKINE proteins, regulate the bioavailability ofNEOKINE proteins, and modulate NEOKINE activity.

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology), c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). A T129 protein interacts with other cellular proteins andcan thus be used for (i) regulation of cellular proliferation; (ii)regulation of cellular differentiation; and (iii) regulation of cellsurvival. The isolated nucleic acid molecules of the invention can beused to express T129 protein (e.g., via a recombinant expression vectorin a host cell in gene therapy applications), to detect T129 mRNA (e.g.,in a biological sample) or a genetic lesion in a T129 gene, and tomodulate T129 activity. In addition, the T129 proteins can be used toscreen drugs or compounds which modulate the T129 activity or expressionas well as to treat disorders characterized by insufficient or excessiveproduction of T129 protein or production of T129 protein forms whichhave decreased or aberrant activity compared to T129 wild type protein.In addition, the anti-T129 antibodies of the invention can be used todetect and isolate T129 proteins and modulate T129 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

FTHMA-070. T85, T129 or A259 Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to FTHMA-070, T85, T129 or A259 proteins or have astimulatory or inhibitory effect on, for example, FTHMA-070, T85, T129or A259 expression or FTHMA-070, T85, T129 or A259 activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of a FTHMA-070, T85, T129 or A259 protein orpolypeptide or biologically active portion thereof. The test compoundsof the present invention can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten (1992) Bio/Techniques13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor(1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores(U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull etal. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scottand Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; and Felici(1991) J. Mol. Biol. 222:301-310).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of FTHMA-070, T85, T129 or A259 protein,or a biologically active portion thereof, on the cell surface iscontacted with a test compound and the ability of the test compound tobind to a FTHMA-070, T85, T129 or A259 protein determined. The cell, forexample, can be a yeast cell or a cell of mammalian origin. Determiningthe ability of the test compound to bind to the FTHMA-070, T85, T129 orA259 protein can be accomplished, for example, by coupling the testcompound with a radioisotope or enzymatic label such that binding of thetest compound to the FTHMA-070, T85, T129 or A259 protein orbiologically active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, test compounds can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product. In apreferred embodiment, the assay comprises contacting a cell whichexpresses a membrane-bound form of FTHMA-070, T85, T129 or A259 protein,or a biologically active portion thereof, on the cell surface with aknown compound which binds FTHMA-070, T85, T129 or A259 to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with aFTHMA-070, T85, T129 or A259 protein, wherein determining the ability ofthe test compound to interact with a FTHMA-070, T85, T129 or A259protein comprises determining the ability of the test compound topreferentially bind to FTHMA-070, T85, T129 or A259 or a biologicallyactive portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of FTHMA-070, T85,T129 or A259 protein, or a biologically active portion thereof, on thecell surface with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe FTHMA-070, T85, T129 or A259 protein or biologically active portionthereof. Determining the ability of the test compound to modulate theactivity of FTHMA-070, T85, T129 or A259 or a biologically activeportion thereof can be accomplished, for example, by determining theability of the FTHMA-070, T85, T129 or A259 protein to bind to orinteract with a FTHMA-070, T85, T129 or A259 target molecule. As usedherein, a “target molecule” is a molecule with which a FTHMA-070, T85,T129 or A259 protein binds or interacts in nature, for example, amolecule on the surface of a cell which expresses a FTHMA-070, T85, T129or A259 protein, a molecule on the surface of a second cell, a moleculein the extracellular milieu, a molecule associated with the internalsurface of a cell membrane or a cytoplasmic molecule. A FTHMA-070, T85,T129 or A259 target molecule can be a non-FTHMA-070, T85, T129 or A259molecule or a FTHMA-070, T85, T129 or A259 protein or polypeptide of thepresent invention. In one embodiment, a FTHMA-070, T85, T129 or A259target molecule is a component of a signal transduction pathway whichfacilitates transduction of an extracellular signal (e.g., a signalgenerated by binding of a compound to a membrane-bound FTHMA-070, T85,T129 or A259 molecule) through the cell membrane and into the cell. Thetarget, for example, can be a second intercellular protein which hascatalytic activity or a protein which facilitates the association ofdownstream signaling molecules with FTHMA-070, T85, T129 or A259.

Determining the ability of the FTHMA-070, T85, T129 or A259 polypeptideof the invention to bind to or interact with a target molecule can beaccomplished by one of the methods described above for determiningdirect binding. As used herein, a “target molecule” is a molecule withwhich a selected polypeptide (e.g., a polypeptide of the invention)binds or interacts with in nature, for example, a molecule on thesurface of a cell which expresses the selected protein, a molecule onthe surface of a second cell, a molecule in the extracellular milieu, amolecule associated with the internal surface of a cell membrane or acytoplasmic molecule. A target molecule can be a FTHMA-070, T85, T129 orA259 polypeptide of the invention or some other polypeptide or protein.For example, a target molecule can be a component of a signaltransduction pathway which facilitates transduction of an extracellularsignal (e.g., a signal generated by binding of a compound to apolypeptide of the invention) through the cell membrane and into thecell or a second intercellular protein which has catalytic activity or aprotein which facilitates the association of downstream signalingmolecules with a polypeptide of the invention. Determining the abilityof a FTHMA-070, T85, T129 or A259 polypeptide of the invention to bindto or interact with a target molecule can be accomplished by determiningthe activity of the target molecule. For example, the activity of thetarget molecule can be determined by detecting induction of a cellularsecond messenger of the target (e.g., intracellular Ca²⁺,diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of areporter gene (e.g., a regulatory element that is responsive to apolypeptide of the invention operably linked to a nucleic acid encodinga detectable marker, e.g., luciferase), or detecting a cellularresponse, for example, cellular differentiation, or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a FTHMA-070, T85, T129 or A259protein or biologically active portion thereof with a test compound anddetermining the ability of the test compound to bind to the FTHMA-070,T85, T129 or A259 protein or biologically active portion thereof.Binding of the test compound to the FTHMA-070, T85, T129 or A259 proteincan be determined either directly or indirectly as described above. In apreferred embodiment, the assay includes contacting the FTHMA-070, T85,T129 or A259 protein or biologically active portion thereof with a knowncompound which binds FTHMA-070, T85, T129 or A259 to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with aFTHMA-070, T85, T129 or A259 protein, wherein determining the ability ofthe test compound to interact with a FTHMA-070, T85, T129 or A259protein comprises determining the ability of the test compound topreferentially bind to FTHMA-070, T85, T129 or A259 or biologicallyactive portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting FTHMA-070, T85, T129 or A259 protein or biologically activeportion thereof with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe FTHMA-070, T85, T129 or A259 protein or biologically active portionthereof. Determining the ability of the test compound to modulate theactivity of FTHMA-070, T85, T129 or A259 can be accomplished, forexample, by determining the ability of the FTHMA-070, T85, T129 or A259protein to bind to a FTHMA-070, T85, T129 or A259 target molecule by oneof the methods described above for determining direct binding. In analternative embodiment, determining the ability of the test compound tomodulate the activity of FTHMA-070, T85, T129 or A259 can beaccomplished by determining the ability of the FTHMA-070, T85, T129 orA259 protein further modulate a FTHMA-070, T85, T129 or A259 targetmolecule. For example, the catalytic/enzymatic activity of the targetmolecule on an appropriate substrate can be determined as previouslydescribed.

In yet another embodiment, the cell-free assay comprises contacting theFTHMA-070, T85, T129 or A259 protein or biologically active portionthereof with a known compound which binds FTHMA-070, T85, T129 or A259to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a FTHMA-070, T85, T129 or A259 protein, wherein determining theability of the test compound to interact with a FTHMA-070, T85, T129 orA259 protein comprises determining the ability of the FTHMA-070, T85,T129 or A259 protein to preferentially bind to or modulate the activityof a FTHMA-070, T85, T129 or A259 target molecule.

In the case of cell-free assays, it may be desirable to utilize asolubilizing agent such that the membrane-bound form of FTHMA-070, T85,T129 or A259 is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either FTHMA-070, T85, T129or A259 or its target molecule to facilitate separation of complexedfrom uncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound toFTHMA-070, T85, T129 or A259, or interaction of FTHMA-070, T85, T129 orA259 with a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example,glutathione-S-transferase/FTHMA-070, T85, T129 or A259 fusion proteinsor glutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or FTHMA-070, T85, T129 or A259 protein, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtitre plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of FTHMA-070, T85, T129 or A259 binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either FTHMA-070,T85, T129 or A259 or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated FTHMA-070, T85,T129 or A259 or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals; Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with. FTHMA-070, T85, T129 or A259 ortarget molecules but which do not interfere with binding of theFTHMA-070, T85, T129 or A259 protein to its target molecule can bederivatized to the wells of the plate, and unbound target or FTHMA-070,T85, T129 or A259 trapped in the wells by antibody conjugation. Methodsfor detecting such complexes, in addition to those described above forthe GST-immobilized complexes, include immunodetection of complexesusing antibodies reactive with the FTHMA-070, T85, T129 or A259 ortarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the FTHMA-070, T85, T129 or A259or target molecule.

In another embodiment, modulators of FTHMA-070, T85, T129 or A259expression are identified in a method in which a cell is contacted witha candidate compound and the expression of FTHMA-070, T85, T129 or A259mRNA or protein in the cell is determined. The level of expression ofFTHMA-070, T85, T129 or A259 mRNA or protein in the presence of thecandidate compound is compared to the level of expression of FTHMA-070,T85, T129 or A259 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof FTHMA-070, T85, T129 or A259 expression based on this comparison. Forexample, when expression of FTHMA-070, T85, T129 or A259 mRNA or proteinis greater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of FTHMA-070, T85, T129 or A259 mRNA orprotein expression. Alternatively, when expression of FTHMA-070, T85,T129 or A259 mRNA or protein is less (statistically significantly less)in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of FTHMA-070, T85, T129or A259 mRNA or protein expression. The level of FTHMA-070, T85, T129 orA259 mRNA or protein expression in the cells can be determined bymethods described herein for detecting FTHMA-070, T85, T129 or A259 mRNAor protein.

In yet another aspect of the invention, the FTHMA-070, T85, T129 or A259proteins can be used as “bait proteins” in a two-hybrid assay or threehybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993)Oncogene 8:1693-1696; and WO94/10300), to identify other proteins, whichbind to or interact with FTHMA-070, T85, T129 or A259 and modulateFTHMA-070, T85, T129 or A259 activity. Such FTHMA-070, T85, T129 orA259-binding proteins are also likely to be involved in the propagationof signals by the FTHMA-070, T85, T129 or A259 proteins as, for example,upstream or downstream elements of the FTHMA-070, T85, T129 or A259pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for FTHMA-070, T85,T129 or A259 is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming an FTHMA-070,T85, T129 or A259-dependent complex, the DNA-binding and activationdomains of the transcription factor are brought into close proximity.This proximity allows transcription of a reporter gene (e.g., LacZ)which is operably linked to a transcriptional regulatory site responsiveto the transcription factor. Expression of the reporter gene can bedetected and cell colonies containing the functional transcriptionfactor can be isolated and used to obtain the cloned gene which encodesthe protein which interacts with FTHMA-070, T85, T129 or A259.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Tango-77 Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to Tango-77 proteins or have a stimulatory or inhibitoryeffect on, for example, Tango-77 expression or Tango-77 activity.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

In another embodiment, an assay is used to determine the ability of thetest compound to modulate the activity of Tango-77 or a biologicallyactive portion thereof, for example, by determining the ability of theTango-77 protein to bind to or interact with a Tango-77 target molecule.As used herein, a “target molecule” is a molecule with which a Tango-77protein binds or interacts in nature, for example, a molecule on thesurface of a cell. A Tango-77 target molecule can be a non-Tango-77molecule or a Tango-77 protein or polypeptide of the present invention.In one embodiment, a Tango-77 target molecule is a component of a signaltransduction pathway, for example, Tango-77 may bind to a IL-1 receptoror another receptor thereby blocking the receptor and inhibiting futuresignal transduction. Determining the ability of the Tango-77 protein tobind to or interact with a Tango-77 target molecule can be accomplishedby one of the methods described above. In a preferred embodiment,determining the ability of the Tango-77 protein to bind to or interactwith a Tango-77 target molecule can be accomplished by determining theactivity of the target molecule. For example, the activity of the targetmolecule can be determined by detecting induction of a cellular secondmessenger of the target (e.g., intracellular Ca²⁺, diacylglycerol, IP3,etc.), detecting catalytic/enzymatic activity of the target on anappropriate substrate, detecting the induction of a reporter gene (e.g.,a Tango-77-responsive regulatory element operably linked to a nucleicacid encoding a detectable marker, e.g. luciferase), or detecting acellular response, for example, inflammation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a Tango-77 protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the Tango-77 protein or biologicallyactive portion thereof. Binding of the test compound to the Tango-77protein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting theTango-77 protein or biologically active portion thereof with a knowncompound which binds Tango-77 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a Tango-77 protein, wherein determiningthe ability of the test compound to interact with a Tango-77 proteincomprises determining the ability of the test compound to preferentiallybind to Tango-77 or biologically active portion thereof as compared tothe known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting Tango-77 protein or biologically active portion thereof witha test compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the Tango-77protein or biologically active portion thereof. Determining the abilityof the test compound to modulate the activity of Tango-77 can beaccomplished, for example, by determining the ability of the Tango-77protein to bind to a Tango-77 target molecule by one of the methodsdescribed above for determining direct binding. In an alternativeembodiment, determining the ability of the test compound to modulate theactivity of Tango-77 can be accomplished by determining the ability ofthe Tango-77 protein to further modulate a Tango-77 target molecule. Forexample, the catalytic/enzymatic activity of the target molecule on anappropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theTango-77 protein or biologically active portion thereof with a knowncompound which binds Tango-77 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a Tango-77 protein, wherein determiningthe ability of the test compound to interact with a Tango-77 proteincomprises determining the ability of the Tango-77 protein topreferentially bind to or modulate the activity of a Tango-77 targetmolecule.

It is possible that membrane-bound forms of Tango-77 exist. Thecell-free assays of the present invention are amenable to use of boththe forms Tango-77. In the case of cell-free assays comprising amembrane-bound form of Tango-77, it may be desirable to utilize asolubilizing agent such that the membrane-bound form of Tango-77 ismaintained in solution. Examples of such solubilizing agents includenon-ionic detergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either Tango-77 or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a test compound to Tango-77, orinteraction of Tango-77 with a target molecule in the presence andabsence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/Tango-77 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical Co.; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or Tango-77 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of Tango-77 binding oractivity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either Tango-77or its target molecule can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated Tango-77 or target molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals;Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withTango-77 or target molecules but which do not interfere with binding ofthe Tango-77 protein to its target molecule can be derivatized to thewells of the plate, and unbound target or Tango-77 trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theTango-77 or target molecule, as well as enzyme-linked assays which relyon detecting an enzymatic activity associated with the Tango-77 ortarget molecule.

In another embodiment, modulators of Tango-77 expression are identifiedin a method in which a cell is contacted with a candidate compound andthe expression of Tango-77 mRNA or protein in the cell is determined.The level of expression of Tango-77 mRNA or protein in the presence ofthe candidate compound is compared to the level of expression ofTango-77 mRNA or protein in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of Tango-77expression based on this comparison. For example, when expression ofTango-77 mRNA or protein is greater (statistically significantlygreater) in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of Tango-77 mRNA orprotein expression. Alternatively, when expression of Tango-77 mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of Tango-77 mRNA or protein expression. Thelevel of Tango-77 mRNA or protein expression in the cells can bedetermined by methods described herein for detecting Tango-77 mRNA orprotein.

In yet another aspect of the invention, the Tango-77 proteins can beused as “bait proteins” in a two-hybrid assay or three hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with Tango-77 (“Tango-77-bindingproteins” or “Tango-77-bp”) and modulate Tango-77 activity. SuchTango-77-binding proteins are also likely to be involved in thepropagation of signals by the Tango-77 proteins as, for example,upstream or downstream elements of the Tango-77 pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for Tango-77 is fusedto a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming an Tango-77-dependent complex,the DNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with Tango-77.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

SPOIL Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to SPOIL proteins or have a stimulatory or inhibitory effecton, for example, SPOIL expression or SPOIL activity and/or have astimulatory or inhibitory effect on IL-1 stimulated activities.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a SPOILtarget molecule. The test compounds of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Mt. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Mt. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries ofcompounds may be presented in solution (e.g., Houghten (1992)Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84),chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No.5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al.(1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith(1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

In one embodiment, the screening assay comprises contacting a cell whichexpresses a SPOIL receptor on the cell surface with a SPOIL protein orbiologically-active portion thereof, to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a SPOIL receptor, whereindetermining the ability of the test compound to interact with a SPOILreceptor comprises determining the ability of the test compound topreferentially bind to the SPOIL receptor as compared to the ability ofSPOIL, or a biologically active portion thereof, to bind to thereceptor. In addition, the screening assay can also comprise contactinga cell which expresses a SPOIL receptor on the cell surface with a SPOILprotein or biological portion thereof, and IL-1, to form a competitivebinding assay. The binding assay can then be contacted with a testcompound in order to determine the ability of the test compound topreferentially bind to the receptor as compared with the SPOIL proteinor biological portion thereof and/or modulate IL-1 stimulated activityby the cell.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a SPOIL target molecule with a testcompound and determining the ability of the test compound to modulate(e.g. stimulate or inhibit) the activity of the SPOIL target molecule.Determining the ability of the test compound to modulate the activity ofa SPOIL target molecule can be accomplished, for example, by determiningthe ability of the SPOIL protein to bind to or interact with the SPOILtarget molecule in the presence of the test compound. This assay can beperformed in the presence of IL-1, and the ability of the SPOIL proteinto interact with the target molecule can be determined by assessing theactivity of a cell that is normally stimulated by IL-1 as compared to acontrol assay comprising cell expressing a SPOIL target molecule, SPOILprotein and IL-1 without the test compound.

Determining the ability of the SPOIL protein to bind to or interact witha SPOIL target molecule can be accomplished by determining the activityof the target molecule. For example, the activity of the target moleculecan be determined by detecting induction or lack of induction of acellular second messenger of the target (i.e. intracellular Ca²⁺,diacylglycerol, IP₃, PGE₂, etc.), detecting catalytic/enzymatic activityof the target an appropriate substrate, detecting the induction of areporter gene (comprising a SPOIL and/or IL-1-responsive regulatoryelement operatively linked to a nucleic acid encoding a detectablemarker, e.g., luciferase), or detecting a cellular response or lack of acellular response, for example, SPOIL and/or IL-1 stimulateddevelopment, differentiation or rate of proliferation.

In yet another embodiment, the assay is a cell-free assay in which aSPOIL protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the SPOIL protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a SPOIL protein can beaccomplished, for example, by determining the ability of the SPOILprotein to bind to a SPOIL target molecule in the presence and/orabsence of the test compound. Determining the ability of the testcompound to modulate the activity of a SPOIL protein can be accomplishedin the presence or absence of IL-1. Determining the ability of the SPOILprotein to bind to a SPOIL target molecule can also be accomplishedusing a technology such as real-time Biomolecular Interaction Analysis(BIA). Sjolander et al. (1991) Anal. Chem. 63:2338-2345 and Szabo et al.(1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore™). Changes in theoptical phenomenon surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either SPOIL or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a SPOIL protein, or interaction ofa SPOIL protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/SPOIL fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or SPOIL protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of SPOILbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a SPOILprotein or a SPOIL target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated SPOIL protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with SPOIL protein or target molecules but which donot interfere with binding of the SPOIL protein to its target moleculecan be derivatized to the wells of the plate, and unbound target orSPOIL protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the SPOIL protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the SPOIL protein or target molecule.

In yet another aspect of the invention, the SPOIL proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with SPOIL (“SPOIL-binding proteins” or “SPOIL-bp”)and modulate SPOIL activity. Such SPOIL-binding proteins are also likelyto be involved in the propagation of signals by the SPOIL proteins as,for example, downstream elements of a SPOIL-mediated signaling pathway.Alternatively, such SPOIL-binding proteins are likely to be cell-surfacemolecules associated with non-SPOIL expressing cells, wherein suchSPOIL-binding proteins are involved in signal transduction.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a SPOIL protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a SPOIL-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the SPOILprotein.

This invention further pertains to novel SPOIL agents such as SPOILproteins or biologically active portions thereof, SPOIL variants whichfunction as IL-1 receptor agonists and nucleic acid molecules encoding aSPOIL protein or variant, which can be screened to determine theefficacy of such agents on various SPOIL and/or IL-1 stimulatedactivities (e.g., stimulated immune response, proliferation, signaltransduction pathway, or differentiation).

In one embodiment, determining the ability of a SPOIL agent to modulatethe activity of SPOIL and/or IL-1 can be accomplished by testing theability of the agent to interfere with the proliferation of T cells inthe presence of SPOIL and/or IL-1.

It is also within the scope of this invention to further use a SPOILagent as described herein in an appropriate animal model. For example,an agent as described herein (e.g., a modulating agent) can be used inan animal model to determine the efficacy, toxicity, or side effects oftreatment with such an agent. Alternatively, a SPOIL agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Animal models for use indetermining the efficacy or mechanism of action of a SPOIL agent of thepresent invention include animal models demonstrating parameters ofsepsis (e.g., animals injected with E. coli to induce hypotension) andanimal models for determining bone metabolism (e.g., lethally irradiatedmice which have been transplanted with SPOIL infected marrow cells).Other animal models which are recognized in the art as predictive ofresults in humans with various IL-1 induced disorders are known in theart and described, for example, in Dinarello (1991) Blood77(8):1627-1652. Furthermore, this invention pertains to uses of SPOILagents and agents identified by the above-described screening assays fortreatments as described herein.

NEOKINE Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to NEOKINE proteins, bind to NEOKINE receptors, have astimulatory or inhibitory effect on, for example, NEOKINE expression,NEOKINE activity, or NEOKINE receptor activity (e.g., RDC1 activity), orhave a stimulatory or inhibitory effect on, for example, the expressionor activity of a non-NEOKINE chemokine or non-NEOKINE chemokinereceptor. It will be appreciated by one of skill in the art thatmodulators identified by the screening assays defined herein (e.g.,modulators of NEOKINE and/or NEOKINE receptor or RDC1) can be used inthe prophylactic and therapeutic treatment of diseases and disordersassociated with aberrant NEOKINE and/or NEOKINE receptor activity (e.g.,proliferative disorders and diseases).

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a NEOKINEprotein or polypeptide or biologically active portion thereof. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of aNEOKINE receptor. The test compounds of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994). J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a NEOKINE receptor on the cell surface is contacted with atest compound and the ability of the test compound to bind to a NEOKINEreceptor determined. The cell, for example, can be of mammalian originor a yeast cell. The NEOKINE receptor can be heterologously expressed orover expressed in a host cell (e.g., a COS cell or fibroblastic cell,for example a HEK293 cell). Alternatively, an assay cell can be selectedwhich endogenously expresses a NEOKINE receptor (e.g., RDC1), forexample, a rat pancreatic acinar cell line, AR4-2J, a PC12pheochromocytoma cell, a SK-N-MC neroblastoma cell, a MES-13 mesangialtumor cell, an astrocyte, or a neutrophil). (Hesen et al. (1998)Immunogenetics 47:364-370 and Law and Rosenzweig (1994) Biochem.Biophys. Res. Commun. 201:458-465). Yeast cells are also particularlyamenable for use in screening assays for G-protein-coupled receptors asdescribed, for example, in Pausch (1997) TIBTECH 15:487-494. Determiningthe ability of the test compound to bind to a NEOKINE receptor can beaccomplished, for example, by coupling the test compound with aradioisotope or enzymatic label such that binding of the test compoundto the NEOKINE receptor can be determined by detecting the labeledcompound in a complex. For example, test compounds can be labeled with¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, test compounds can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a test compound to interact with an NEOKINE receptor without thelabeling of any of the interactants. For example, a microphysiometer canbe used to detect the interaction of a test compound with an NEOKINEreceptor without the labeling of either the test compound or thereceptor. McConnell, H. M. et al. (1992) Science 257:1906-1912. As usedherein, a “microphysiometer” (e.g., Cytosensor™) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between ligand and receptor.

In a preferred embodiment, the assay comprises contacting a cell whichexpresses an NEOKINE receptor on the cell surface with a NEOKINE proteinor biologically-active portion thereof and a test compound, anddetermining the ability of the test compound to modulate binding of theNEOKINE protein or biologically-active portion thereof to the NEOKINEreceptor. Determining the ability of the test compound to modulatebinding of the NEOKINE protein or biologically-active portion thereof tothe NEOKINE receptor can comprise determining the ability of the testcompound to preferentially bind to the NEOKINE receptor as compared tothe ability of NEOKINE, or a biologically active portion thereof, tobind to the receptor. Alternatively, determining the ability of the testcompound to modulate binding of the NEOKINE protein orbiologically-active portion thereof to the NEOKINE receptor can comprisedetermining a change in the binding of the NEOKINE protein orbiologically-active portion thereof to the NEOKINE receptor (e.g., achange in the amount of binding in the presence of the test compound ascompared to the absence of the test compound).

In another preferred embodiment, the assay comprises contacting a cellwhich expresses a receptor specific for another chemokine on the cellsurface with an NEOKINE protein or biologically-active portion thereofand a test compound, and determining the ability of the test compound tointeract with the receptor, wherein determining the ability of the testcompound to interact with the receptor comprises determining the abilityof the test compound to preferentially bind to the receptor as comparedto the ability of the NEOKINE, or a biologically active portion thereof,to bind to the receptor. Alternatively, determining the ability of thetest compound to interact with the NEOKINE receptor can comprisedetermining a change in the binding of the NEOKINE protein orbiologically-active portion thereof to the NEOKINE receptor (e.g., achange in the amount of binding).

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a NEOKINE target molecule (e.g. a NEOKINEreceptor) with a test compound and determining the ability of the testcompound to modulate (e.g. stimulate or inhibit) the activity of theNEOKINE target molecule. In yet another embodiment, an assay is acell-based assay comprising contacting a cell expressing a NEOKINEreceptor with a NEOKINE protein or biologically-active portion thereofand a test compound and determining the ability of the test compound tomodulate the activity of the NEOKINE target molecule.

Determining the ability of the NEOKINE protein to bind to or interactwith an NEOKINE target molecule can be accomplished by one of themethods described above for determining direct binding. The activity ofthe target molecule can be determined by detecting induction of acellular second messenger of the target (i.e. intracellular Ca²⁺,diacylglycerol, IP₃, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of areporter gene (comprising an NEOKINE-responsive regulatory elementoperatively linked to a nucleic acid encoding a detectable marker, e.g.,luciferase), or detecting a cellular response, for example, anangiogenic response or an inflammatory response.

In yet another embodiment, an assay of the present invention is acell-free assay in which an NEOKINE protein or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to bind to the NEOKINE protein or biologically activeportion thereof is determined. Binding of the test compound to theNEOKINE protein can be determined either directly or indirectly asdescribed above. In a preferred embodiment, the assay includescontacting the NEOKINE protein or biologically active portion thereofwith a known compound which binds NEOKINE to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with an NEOKINE protein,wherein determining the ability of the test compound to interact with anNEOKINE protein comprises determining the ability of the test compoundto preferentially bind to NEOKINE or biologically active portion thereofas compared to the known compound.

In another embodiment, the assay is a cell-free assay in which anNEOKINE protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the NEOKINE protein orbiologically active portion thereof is determined. Determining theability of the test compound to modulate the activity of an NEOKINEprotein can be accomplished, for example, by determining the ability ofthe NEOKINE protein to bind to an NEOKINE target molecule by one of themethods described above for determining direct binding. Determining theability of the NEOKINE protein to bind to an NEOKINE target molecule canalso be accomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander and Urbaniczky (1991) Anal. Chem.63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705. As used herein, “BIA” is a to technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore™). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of an NEOKINE protein can beaccomplished by determining the ability of the NEOKINE protein tofurther modulate the activity of a downstream effector (e.g., anintracellular signaling molecule) of an NEOKINE target molecule (e.g.,an NEOKINE receptor). For example, the catalytic/enzymatic activity ofthe effector molecule on an appropriate substrate can be determined aspreviously described.

In yet another embodiment, the cell-free assay involves contacting anNEOKINE protein or biologically active portion thereof with a knowncompound which binds the NEOKINE protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the NEOKINE protein,wherein determining the ability of the test compound to interact withthe NEOKINE protein comprises determining the ability of the NEOKINEprotein to preferentially bind to or modulate the activity of an NEOKINEtarget molecule.

The assays of the present invention are based at least in part on thediscovery that NEOKINE receptor is the previously identified orphanchemokine receptor, RDC1. The nucleic acid sequence of human, murine andcanine RDC1 are set forth in SEQ ID NO:129, SEQ ID NO:131 and SEQ IDNO:133, respectively. The amino acid sequences of human, murine andcanine RDC1 are set forth in SEQ ID NO:130, SEQ ID NO:132 or SEQ IDNO:134, respectively. Human, murine and canine RDC1 sequences can befurther found at Accession Nos. U73141 & U67784, AF000236, and X14048,respectively. Accordingly, in one embodiment, the NEOKINE receptor hasthe amino acid set forth in any of SEQ ID NO:130, SEQ ID NO:132 or SEQID NO:134. In another embodiment, the NEOKINE receptor is selectedencoded by a nucleic acid molecule selected from the group consisting ofSEQ ID NO:129, SEQ ID NO:131 or SEQ ID NO:133. In another embodiment theNEOKINE receptor is selected from the group consisting of a receptorhaving an amino acid sequence which is substantially homologous to theamino acid sequence of any of SEQ ID NO:130, SEQ ID NO:132 or SEQ IDNO:134; a receptor which is encoded by an isolated nucleic acid moleculewhich is substantially homologous to any of SEQ ID NO:129, SEQ ID NO:131or SEQ ID NO:133; or a receptor which is encoded by an isolated nucleicacid molecule which hybridizes under stringent conditions to a nucleicacid molecule having any of SEQ ID NO:129, SEQ ID NO:131 or SEQ IDNO:133.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins (e.g.NEOKINE proteins or biologically active portions thereof or NEOKINEreceptors). In the case of cell-free assays in which a membrane-boundform an isolated protein is used (e.g., a NEOKINE receptor) it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the isolated protein is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either NEOKINE or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a test compound to a NEOKINEprotein, or interaction of a NEOKINE protein with a target molecule inthe presence and absence of a candidate compound, can be accomplished inany vessel suitable for containing the reactants. Examples of suchvessels include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/NEOKINE fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or NEOKINE protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofNEOKINE binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a NEOKINEprotein or a NEOKINE target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated NEOKINE protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with NEOKINE protein or target molecules but whichdo not interfere with binding of the NEOKINE protein to its targetmolecule can be derivatized to the wells of the plate, and unboundtarget or NEOKINE protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the NEOKINE protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the NEOKINE protein or targetmolecule.

In another embodiment, modulators of NEOKINE expression are identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of NEOKINE mRNA or protein in the cell is determined. Thelevel of expression of NEOKINE mRNA or protein in the presence of thecandidate compound is compared to the level of expression of NEOKINEmRNA or protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of NEOKINE expressionbased on this comparison. For example, when expression of NEOKINE mRNAor protein is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of NEOKINE mRNA or proteinexpression. Alternatively, when expression of NEOKINE mRNA or protein isless (statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of NEOKINE mRNA or protein expression. The level of NEOKINEmRNA or protein expression in the cells can be determined by methodsdescribed herein for detecting NEOKINE mRNA or protein.

In yet another aspect of the invention, the NEOKINE proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with NEOKINE (“NEOKINE-binding proteins” or“NEOKINE-bp”) and are involved in NEOKINE activity. Such NEOKINE-bindingproteins are also likely to be involved in the propagation of signals bythe NEOKINE proteins as, for example, downstream elements of aNEOKINE-mediated signaling pathway. Alternatively, such NEOKINE-bindingproteins are likely to be cell-surface molecules associated withnon-NEOKINE expressing cells, wherein such NEOKINE-binding proteins areinvolved in chemoattraction.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a NEOKINE proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aNEOKINE-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the NEOKINE protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a NEOKINE modulating agent, an antisense NEOKINEnucleic acid molecule, a NEOKINE-specific antibody, or a NEOKINE-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 DetectionAssays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 ChromosomeMapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 nucleic acid molecules described herein or fragmentsthereof, can be used to map the location of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 genes on a chromosome. The mapping ofthe FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259sequences to chromosomes is an important first step in correlating thesesequences with genes associated with disease.

Briefly, FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259genes can be mapped to chromosomes by preparing PCR primers (preferably15-25 bp in length) from the FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 sequences. Computer analysis of FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequences can be usedto rapidly select primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primerscan then be used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio et al. (1983) Science220:919-924). Somatic cell hybrids containing only fragments of humanchromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using theFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequencesto design oligonucleotide primers, sublocalization can be achieved withpanels of fragments from specific chromosomes. Other mapping strategieswhich can similarly be used to map a FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 sequence to its chromosome include in situhybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide, a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York, 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature,325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene can be determined. If amutation is observed in some or all of the affected individuals but notin any unaffected individuals, then the mutation is likely to be thecausative agent of the particular disease. Comparison of affected andunaffected individuals generally involves first looking for structuralalterations in the chromosomes such as deletions or translocations thatare visible from chromosome spreads or detectable using PCR based onthat DNA sequence. Ultimately, complete sequencing of genes from severalindividuals can be performed to confirm the presence of a mutation andto distinguish mutations from polymorphisms.

2. FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 TissueTyping

The FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 sequences described herein can be used to prepare two PCRprimers from the 5′ and 3′ ends of the sequences. These primers can thenbe used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259sequences of the invention uniquely represent portions of the humangenome. Allelic variation occurs to some degree in the coding regions ofthese sequences, and to a greater degree in the noncoding regions. It isestimated that allelic variation between individual humans occurs with afrequency of about once per each 500 bases. Each of the sequencesdescribed herein can, to some degree, be used as a standard againstwhich DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:53, 57, 71, 89, 101,16, 112, 115, 137, 145 or 163 can comfortably provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:55, 59, 73, 76,80, 91, 103, 106, 117, 139, 146 or 164 are used, a more appropriatenumber of primers for positive individual identification would be500-2,000.

If a panel of reagents from FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 sequences described herein is used to generate a uniqueidentification database for an individual, those same reagents can laterbe used to identify tissue from that individual. Using the uniqueidentification database, positive identification of the individual,living or dead, can be made from extremely small tissue samples.

3. Use of Partial FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the FTHMA-070, T85 or A259sequences or portions thereof, e.g., fragments derived from thenoncoding regions having a length of at least 20 or 30 bases.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:71, SEQ ID NO:89, SEQ IDNO:101, or SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:115 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theTango77, SPOIL, NEOKINE or Tango129 nucleotide sequences or portionsthereof, e.g., fragments derived from the noncoding regions of SEQ IDNO:71, SEQ ID NO:89, SEQ ID NO:101, or SEQ ID NO:104, SEQ ID NO:112, SEQID NO:115 having a length of at least 20 bases, preferably at least 30bases.

The FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259sequences described herein can further be used to provide polynucleotidereagents, e.g., labeled or labelable probes which can be used in, forexample, an in situ hybridization technique, to identify a specifictissue, e.g., brain tissue. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 probes can be used to identify tissue by species and/or by organtype.

In a similar fashion, these reagents, e.g., FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 primers or probes can be used to screentissue culture for contamination (i.e., screen for the presence of amixture of different types of cells in a culture).

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 PredictiveMedicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteinand/or nucleic acid expression as well as FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 activity, in the context of abiological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrantFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 expressionor activity. The invention also provides for prognostic (or predictive)assays for determining whether an individual is at risk of developing adisorder associated with FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein, nucleic acid expression or activity. Forexample, mutations in a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene can be assayed in a biological sample. Such assayscan be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein, nucleic acid expression or activity.

Another aspect of the invention provides methods for determiningFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein,nucleic acid expression or FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 activity in an individual to thereby select appropriatetherapeutic or prophylactic agents for that individual (referred toherein as “pharmacogenomics”). Pharmacogenomics allows for the selectionof agents (e.g., drugs) for therapeutic or prophylactic treatment of anindividual based on the genotype of the individual (e.g., the genotypeof the individual examined to determine the ability of the individual torespond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 inclinical trials.

These and other agents are described in further detail in the followingsections.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 DiagnosticAssays

An exemplary method for detecting the presence or absence of FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 in a biologicalsample involves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein or nucleic acid (e.g., mRNA, genomic DNA) that encodesFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 proteinsuch that the presence of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 is detected in the biological sample. A preferred agentfor detecting FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 mRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 mRNA or genomic DNA. The nucleic acid probe can be, for example, afull-length FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango 129 orA259 nucleic acid, such as the nucleic acid of SEQ ID NO:53, 55, 57, 58,71, 73, 76, 80, 89, 101, 104, 112, 115, 118, 121, 124, 137, 139, 145,146, 163 or 164, or the DNA insert of the plasmid deposited with ATCC asAccession Number 98751, or a portion thereof, such as an oligonucleotideof at least 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions toFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein is an antibody capable of binding toFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 mRNA, protein, or genomic DNA in a biological sample in vitro aswell as in vivo. For example, in vitro techniques for detection ofFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Invitro techniques for detection of FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 protein include introducinginto a subject a labeled anti-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 protein, mRNA, or genomic DNA,such that the presence of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein, mRNA or genomic DNA is detected in thebiological sample, and comparing the presence of FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 protein, mRNA or genomic DNAin the control sample with the presence of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein, mRNA or genomic DNA in thetest sample.

The invention also encompasses kits for detecting the presence ofFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 in abiological sample (a test sample). Such kits can be used to determine ifa subject is suffering from or is at increased risk of developing adisorder associated with aberrant expression of FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 (e.g., an immunologicaldisorder or proliferative disorder, e.g., psoriasis or cancer). Forexample, the kit can comprise a labeled compound or agent capable ofdetecting FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein or mRNA in a biological sample and means for determining theamount of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259in the sample (e.g., an anti-FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 antibody or an oligonucleotide probe whichbinds to DNA encoding FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259). Kits may also include instruction for observing thatthe tested subject is suffering from or is at risk of developing adisorder associated with aberrant expression of FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 if the amount of FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein or mRNA isabove or below a normal level.

For antibody-based kits, the kit may comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein; and,optionally, (2) a second, different antibody which binds to FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein or the firstantibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit may comprise, for example: (1) aoligonucleotide, e.g., a detectably labelled oligonucleotide, whichhybridizes to a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 nucleic acid sequence or (2) a pair of primers useful foramplifying a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 nucleic acid molecule;

The kit may also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit may also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit may also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango 129 or A259.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 PrognosticAssays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant FTHMA-070,Tango85, Tango77 or Tango129 expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with FTHMA-070, Tango85,Tango77 or Tango129 protein, nucleic acid expression or activity such asan immune system disorder. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing such adisease or disorder. Thus, the present invention provides a method inwhich a test sample is obtained from a subject and FTHMA-070, Tango85,Tango77 or Tango129 protein or nucleic acid (e.g., mRNA, genomic DNA) isdetected, wherein the presence of FTHMA-070, Tango85, Tango77 orTango129 protein or nucleic acid is diagnostic for a subject having orat risk of developing a disease or disorder associated with aberrantFTHMA-070, Tango85, Tango77 or Tango129 expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant SPOIL and/or IL-1 expression or activity. Forexample, the assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with aberrantSPOIL protein, nucleic acid expression or activity and/or characterizedby aberrant IL-1 expression or activity such as an inflammatorydisorder, an immune disorder, or a differentiative disorder (e.g., abone metabolism disorder). Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adifferentiative or proliferative disease (e.g., leukemia), aninflammatory disease, or an immune disease. Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant SPOIL and/or IL-1 expression or activity in which a test sampleis obtained from a subject and SPOIL protein or nucleic acid (e.g, mRNA,genomic DNA) is detected, wherein the presence of SPOIL protein ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder characterized aberrant SPOIL and/or IL-1expression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant NEOKINE expression or activity. For example,the assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with NEOKINE protein, nucleicacid expression or activity such as an inflammatory disorder.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing an inflammatory disorder. Thus,the present invention provides a method for identifying a disease ordisorder associated with aberrant NEOKINE expression or activity inwhich a test sample is obtained from a subject and NEOKINE protein ornucleic acid (e.g, mRNA, genomic DNA) is detected, wherein the presenceof NEOKINE protein or nucleic acid is diagnostic for a subject having orat risk of developing a disease or disorder associated with aberrantNEOKINE expression or activity. As used herein, a “test sample” refersto a biological sample obtained from a subject of interest. For example,a test sample can be a biological fluid (e.g., serum), cell sample, ortissue.

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of a A259 polypeptide of the invention. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with aberrant expression oractivity of a A259 polypeptide of the invention, e.g., an immunologicdisorder, e.g., asthma, anaphylaxis, or atopic dermatitis.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing such a disease or disorder.Thus, the present invention provides a method in which a test sample isobtained from a subject and a A259 polypeptide or nucleic acid (e.g.,mRNA, genomic DNA) of the invention is detected, wherein the presence ofthe A259 polypeptide or nucleic acid is diagnostic for a subject havingor at risk of developing a disease or disorder associated with aberrantexpression or activity of the A259 polypeptide. As used herein, a “testsample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 expression or activity. For example, such methods canbe used to determine whether a subject can be effectively treated with aspecific agent or class of agents (e.g., agents of a type which decreaseFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 activity).Thus, the present invention provides methods for determining whether asubject can be effectively treated with an agent for a disorderassociated with aberrant FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 expression or activity in which a test sample isobtained and FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 protein or nucleic acid is detected (e.g., wherein the presence ofFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein ornucleic acid is diagnostic for a subject that can be administered theagent to treat a disorder associated with aberrant FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 expression or activity).

The methods of the invention can also be used to detect genetic lesionsor mutations in a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 gene, thereby determining if a subject with the lesioned gene isat risk for a disorder characterized by aberrant cell proliferationand/or differentiation. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by at least one of analteration affecting the integrity of a gene encoding a FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259-protein, or themis-expression of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene. For example, such genetic lesions can be detectedby ascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene; 2) an addition of one or more nucleotides to aFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene; 3) asubstitution of one or more nucleotides of a FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene, 4) a chromosomalrearrangement of a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259 gene; 5) an alteration in the level of a messenger RNAtranscript of a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 gene, 6) aberrant modification of a FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 gene, such as of the methylationpattern of the genomic DNA, 7) the presence of a non-wild type splicingpattern of a messenger RNA transcript of a FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango 129 or A259 gene, 8) a non-wild type level of aFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259-protein,9) allelic loss of a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene, and 10) inappropriate post-translationalmodification of a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129or A259-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in aFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 gene. Apreferred biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Nail. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259-gene (see Abravaya etal. (1995) Nucleic Acids Res. 23:675-682). This method can include thesteps of collecting a sample of cells from a patient, isolating nucleicacid (e.g., genomic, mRNA or both) from the cells of the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 gene under conditions such that hybridization andamplification of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259-gene (if present) occurs, and detecting the presence orabsence of an amplification product, or detecting the size of theamplification product and comparing the length to a control sample. Itis anticipated that PCR and/or LCR may be desirable to use as apreliminary amplification step in conjunction with any of the techniquesused for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene from a sample cell can beidentified by alterations in restriction enzyme cleavage patterns. Forexample, sample and control DNA is isolated, amplified (optionally),digested with one or more restriction endonucleases, and fragment lengthsizes are determined by gel electrophoresis and compared. Differences infragment length sizes between sample and control DNA indicates mutationsin the sample DNA. Moreover, the use of sequence specific ribozymes(see, e.g., U.S. Pat. No. 5,498,531) can be used to score for thepresence of specific mutations by development or loss of a ribozymecleavage site.

In other embodiments, genetic mutations in FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 can be identified by hybridizing asample and control nucleic acids, e.g., DNA or RNA, to high densityarrays containing hundreds or thousands of oligonucleotides probes(Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996)Nature Medicine 2:753-759). For example, genetic mutations in FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 can be identified intwo-dimensional arrays containing light-generated DNA probes asdescribed in Cronin et al. supra. Briefly, a first hybridization arrayof probes can be used to scan through long stretches of DNA in a sampleand control to identify base changes between the sequences by makinglinear arrays of sequential overlapping probes. This step allows theidentification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango 129 or A259 gene and detectmutations by comparing the sequence of the sample FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 with the correspondingwild-type (control) sequence. Examples of sequencing reactions includethose based on techniques developed by Maxim and Gilbert ((1977) Proc.Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci.USA 74:5463). It is also contemplated that any of a variety of automatedsequencing procedures can be utilized when performing the diagnosticassays ((1995) Bio/Techniques 19:448), including sequencing by massspectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al.(1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl.Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene include methods in whichprotection from cleavage agents is used to detect mismatched bases inRNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 sequence with potentially mutant RNA or DNAobtained from a tissue sample. The double-stranded duplexes are treatedwith an agent which cleaves single-stranded regions of the duplex suchas which will exist due to basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digestingthe mismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,e.g., Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 cDNAs obtained from samples of cells.For example, the mutY enzyme of E. coli cleaves A at G/A mismatches andthe thymidine DNA glycosylase from HeLa cells cleaves T at G/Tmismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According toan exemplary embodiment, a probe based on a FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 sequence, e.g., a wild-type FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 sequence, ishybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike. See, e.g., U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 genes. For example, single strand conformationpolymorphism (SSCP) may be used to detect differences in electrophoreticmobility between mutant and wild type nucleic acids (Orita et al. (1989)Proc Natl. Acad. Sci. USA: 86:2766, see also Cotton (1993) Mutat. Res.285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).Single-stranded DNA fragments of sample and control FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 nucleic acids will bedenatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a FTHMA-070, Tango85,Tango77, SPOIL, NEOKINE, Tango129 or A259 gene.

Furthermore, any cell type or tissue, e.g., chondrocytes, or peripheralblood leukocytes, in which FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 is expressed may be utilized in the prognostic assaysdescribed herein.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onFTHMA-070, T85 or A259 activity (e.g., FTHMA-070, T85 or A259 geneexpression) as identified by a screening assay described herein can beadministered to individuals to treat (prophylactically ortherapeutically) disorders (e.g., an immunological disorder) associatedwith aberrant FTHMA-070, T85 or A259 activity. Agents, or modulatorswhich have a stimulatory or inhibitory effect on Tango-77 activity(e.g., Tango-77 gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g., acute or chronicinflammation and asthma) associated with aberrant Tango-77 activity. TheSPOIL molecules of the present invention or SPOIL modulators asidentified by a screening assay described herein can be administered toindividuals to treat (prophylactically or therapeutically) disorders(e.g, inflammatory or developmental disorders) associated with aberrantSPOIL and/or IL-1 activity. The NEOKINE molecules of the presentinvention, as well as agents, or modulators which have a stimulatory orinhibitory effect on NEOKINE activity (e.g., NEOKINE gene expression) asidentified by a screening assay described herein can be administered toindividuals to treat (prophylactically or therapeutically) disorders(e.g, inflammatory disorders) associated with aberrant NEOKINE activity.Agents, or modulators which have a stimulatory or inhibitory effect onT129 activity (e.g., T129 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) disorders (e.g., an immunologicaldisorder) associated with aberrant T129 activity. In conjunction withsuch treatment, the pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) of the individual may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 protein, expression of FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 nucleic acid, ormutation content of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug target is known (e.g., a SPOIL orNEOKINE protein or SPOIL or NEOKINE receptor of the present invention),all common variants of that gene can be identified in the population anda particular drug response can be associated with one or more genes.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a SPOIL or NEOKINEmolecule or SPOIL or NEOKINE modulator of the present invention)indicate whether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a SPOIL or NEOKINEmolecule or SPOIL or NEOKINE modulator, such as a modulator identifiedby one of the exemplary screening assays described herein.

Thus, the activity of FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 protein, expression of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 nucleic acid, or mutation content ofFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 genes inan individual can be determined to thereby select appropriate agent(s)for therapeutic or prophylactic treatment of the individual. Inaddition, pharmacogenetic studies can be used to apply genotyping ofpolymorphic alleles encoding drug-metabolizing enzymes to theidentification of an individual's drug responsiveness phenotype. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 modulator, such as amodulator identified by one of the exemplary screening assays describedherein.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 Monitoringof Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of FTHMA-070 or T85 (e.g., the ability tomodulate aberrant cell proliferation and/or differentiation) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay as described herein to increase FTHMA-070 or T85 gene expression,protein levels, or upregulate FTHMA-070 or T85 activity, can bemonitored in clinical trails of subjects exhibiting decreased FTHMA-070or T85 gene expression, protein levels, or downregulated FTHMA-070 orT85 activity. Alternatively, the effectiveness of an agent determined bya screening assay to decrease FTHMA-070 or T85 gene expression, proteinlevels, or downregulated FTHMA-070 or T85 activity, can be monitored inclinical trails of subjects exhibiting increased FTHMA-070 or T85 geneexpression, protein levels, or upregulated FTHMA-070 or T85 activity. Insuch clinical trials, the expression or activity of FTHMA-070 or T85and, preferably, other genes that have been implicated in, for example,a cellular proliferation disorder can be used as a “read out” or markersof the immune responsiveness of a particular cell.

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of Tango-77 (e.g., the ability to modulateaberrant inflammation) can be applied not only in basic drug screening,but also in clinical trials. For example, the effectiveness of an agent,as determined by a screening assay as described herein, to increaseTango-77 gene expression, increase protein levels, or upregulateTango-77 activity, can be monitored in clinical trials of subjectsexhibiting decreased Tango-77 gene expression, decreased protein levels,or downregulated Tango-77 activity. Alternatively, the effectiveness ofan agent, as determined by a screening assay, to decrease Tango-77 geneexpression, decrease protein levels, or downregulate Tango-77 activity,can be monitored in clinical trials of subjects exhibiting increasedTango-77 gene expression, increased protein levels, or upregulatedTango-77 activity.

Monitoring the influence of SPOIL agents (e.g., modulatory agents and/orSPOIL proteins) on the expression or activity of SPOIL and/or IL-1(e.g., modulation of signal transduction, modulation of cell developmentor differentiation, regulation of cellular proliferation) can be appliednot only in basic drug screening, but also in clinical trials. Forexample, the effectiveness of an agent determined by a screening assay(as described herein) to modulate SPOIL and/or IL-1 expression oractivity can be monitored in clinical trails of subjects exhibitingincreased SPOIL and/or IL-1 expression or activity and/or decreasedSPOIL and/or IL-1 gene expression, protein levels or activity.Alternatively, the effectiveness of an agent determined by a screeningassay to increase SPOIL and/or IL-1 expression or activity and/ordownregulate SPOIL and/or IL-1 gene expression, protein levels oractivity, can be monitored in clinical trails of subjects exhibitingincreased SPOIL and/or IL-1 expression or activity and/or decreasedSPOIL and/or IL-1 gene expression, protein levels or activity. In suchclinical trials, the expression or activity of SPOIL and/or IL-1 and,preferably, other genes that have been implicated in, for example, aproinflammatory disorder, an immune disorder, or a bone metabolismdisorder can be used as a “read out” or markers of the phenotype of aparticular cell.

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of a NEOKINE protein (e.g., modulation ofangiogenesis or of an inflammatory response) an be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase NEOKINE gene expression, protein levels, orupregulate NEOKINE activity, can be monitored in clinical trials ofsubjects exhibiting decreased NEOKINE gene expression, protein levels,or downregulated NEOKINE activity. Alternatively, the effectiveness ofan agent determined by a screening assay to decrease NEOKINE geneexpression, protein levels, or downregulate NEOKINE activity, can bemonitored in clinical trials of subjects exhibiting increased NEOKINEgene expression, protein levels, or upregulated NEOKINE activity. Insuch clinical trials, the expression or activity of a NEOKINE gene, andpreferably, other genes that have been implicated in, for example, aninflammatory disorder can be used as a “read out” or markers of thephenotype of a particular cell.

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of T129 or A259 (e.g., the ability to modulateaberrant cell proliferation and/or differentiation) can be applied notonly in basic drug screening, but also in clinical trials. For example,the effectiveness of an agent determined by a screening assay asdescribed herein to increase T129 or A259 gene expression, proteinlevels, or upregulate T129 or A259 activity, can be monitored inclinical trails of subjects exhibiting decreased T129 or A259 geneexpression, protein levels, or downregulated T129 or A259 activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease T129 or A259 gene expression, protein levels, ordownregulated T129 or A259 activity, can be monitored in clinical trailsof subjects exhibiting increased T129 or A259 gene expression, proteinlevels, or upregulated T129 or A259 activity. In such clinical trials,the expression or activity of T129 or A259 and, preferably, other genesthat have been implicated in, for example, a cellular proliferationdisorder can be used as a “read out” or markers of the immuneresponsiveness of a particular cell.

For example, and not by way of limitation, genes, including FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259, that are modulatedin cells by treatment with an agent (e.g., compound, drug or smallmolecule) which modulates FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE,Tango129 or A259 activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson cellular proliferation disorders, proinflammatory disorders, ordevelopmental or differentiative disorders (e.g., a bone metabolismdisorder) for example, in a clinical trial, cells can be isolated andRNA prepared and analyzed for the levels of expression of FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 protein, mRNA, orgenomic DNA in the preadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 protein, mRNA, or genomic DNA in thepost-administration samples; (v) comparing the level of expression oractivity of the FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 protein, mRNA, or genomic DNA in the pre-administration sample withthe FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259protein, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of FTHMA-070, Tango85, Tango77, SPOIL,NEOKINE, Tango129 or A259 to lower levels than detected, i.e., todecrease the effectiveness of the agent.

FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 Methods ofTreatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant FTHMA-070, Tango85, SPOILor NEOKINE expression or activity.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) developingor having a disorder associated with aberrant Tango-77 expression oractivity. Alternatively, disorders associated with aberrant IL-1production can be treated with Tango-77. Such disorders include acuteand chronic inflammation, asthma, some classes of arthritis, autoimmunediabetes, systemic lupus erythematosus and inflammatory bowel disease.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant T129 expression oractivity. Such disorders include immunological disorders, e.g.,disorders associated with abnormal lymphoid and/or thymic development,T-cell mediated immune response, T-cell dependent help for B cells, andabnormal humoral B cell activity, and, possibly, disorders of theskeletal muscle.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant A259 expression oractivity of a polypeptide of the invention. For example, disorderscharacterized by aberrant A259 expression or activity of thepolypeptides of the invention include immunologic disorders. Inaddition, the A259 nucleic acids, polypeptides, and modulators thereofof the invention can be used to treat immunologic diseases anddisorders, including but not limited to inflammatory disorders (e.g.,atopic dermatitis). A259 polypeptides of the invention can also treatdiseases associated and bone and cartilage degenerative diseases anddisorders (e.g., arthritis, e.g., rheumatoid arthritis), as well asother disorders described herein.

Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 expression oractivity, by administering to the subject an agent which modulatesFTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 expressionor at least one FTHMA-070, Tango85, Tango77, SPOIL, NEOKINE, Tango129 orA259 activity. Subjects at risk for a disease which is caused orcontributed to by aberrant FTHMA-070, Tango85, Tango77, NEOKINE,Tango129 or A259 expression or activity can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 aberrancy, such thata disease or disorder is prevented or, alternatively, delayed in itsprogression. Depending on the type of FTHMA-070, Tango85, Tango77,SPOIL, NEOKINE, Tango129 or A259 aberrancy, for example, a FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 agonist or FTHMA-070,Tango85, Tango77, SPOIL, NEOKINE, Tango129 or A259 antagonist agent canbe used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein. For example, anantagonist of an A259 protein may be used to treat an arthropathicdisorder, e.g., rheumatoid arthritis. The appropriate agent can bedetermined based on screening assays described herein.

Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingFTHMA-070, Tango85, Tango77, Tango129 or A259 expression or activity fortherapeutic purposes. The modulatory method of the invention involvescontacting a cell with an agent that modulates one or more of theactivities of FTHMA-070, Tango85, Tango77, Tango129 or A259 proteinactivity associated with the cell. An agent that modulates FTHMA-070,Tango85, Tango77, Tango129 or A259 protein activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a FTHMA-070, Tango85, Tango77,Tango129 or A259 protein, a peptide, a FTHMA-070, Tango85, Tango77,Tango129 or A259 peptidomimetic, or other small molecule. In oneembodiment, the agent stimulates one or more of the biologicalactivities of FTHMA-070, Tango85, Tango77, Tango129 or A259 protein.Examples of such stimulatory agents include active FTHMA-070, Tango85,Tango77, Tango129 or A259 protein and a nucleic acid molecule encodingFTHMA-070, Tango85, Tango77, Tango129 or A259 that has been introducedinto the cell. In another embodiment, the agent inhibits one or more ofthe biological activities of FTHMA-070, Tango85, Tango77, Tango129 orA259 protein. Examples of such inhibitory agents include antisenseFTHMA-070, Tango85, Tango77, Tango129 or A259 nucleic acid molecules andanti-FTHMA-070, Tango85, Tango77, Tango129 or A259 antibodies. Thesemodulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g, by administeringthe agent to a subject). As such, the present invention provides methodsof treating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a FTHMA-070,Tango85, Tango77, Tango129 or A259 protein or nucleic acid molecule. Inone embodiment, the method involves administering an agent (e.g., anagent identified by a screening assay described herein), or combinationof agents that modulates (e.g., upregulates or down-regulates)FTHMA-070, Tango85, Tango77, Tango129 or A259 expression or activity. Inanother embodiment, the method involves administering a FTHMA-070,Tango85, Tango77, Tango129 or A259 protein or nucleic acid molecule astherapy to compensate for reduced or aberrant FTHMA-070, Tango85,Tango77, Tango129 or A259 expression or activity.

Another aspect of the invention pertains to methods of modulating SPOILand/or IL-1 expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of SPOIL and/or IL-1associated with the cell or one or more of the activities involved ininflammation, immune response, or bone turnover. An agent that modulatesSPOIL and/or IL-1 activity can be an agent as described herein, such asa SPOIL modulator, a nucleic acid encoding a SPOIL protein or a SPOILprotein, a SPOIL peptide, or SPOIL peptidomimetic. In one embodiment,the agent stimulates one or more SPOIL and/or IL-1 protein activity.Examples of such stimulatory agents include SPOIL variants which haveSPOIL receptor and/or IL-1 receptor agonist function or a nucleic acidmolecule encoding such a SPOIL variant that has been introduced into acell. In another embodiment, the agent inhibits one or more SPOIL and/orIL-1 activity. Examples of such inhibitory agents include SPOIL proteinsand nucleic acid molecules, mutant SPOIL proteins and nucleic acidmolecules, antisense SPOIL nucleic acid molecules and SPOIL antibodies.These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant SPOIL and/or IL-1 expression oractivity. In one embodiment, the method involves administering a SPOILagent (e.g., an agent described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) SPOIL and/or IL-1expression or activity. In another embodiment, the method involvesadministering a SPOIL protein or nucleic acid molecule as therapy tocompensate for reduced SPOIL expression or activity.

Another aspect of the invention pertains to methods of modulatingNEOKINE or chemokine expression or activity for therapeutic purposes. Ithas been determined that NEOKINE-1 is strongly expressed in the kidney.This expression of NEOKINE-1 indicates that the NEOKINES have utility intreating kidney inflammation, a major cause of renal failure in chronicand acute renal failure and transplantation. It is known in the art thatexpression of chemokines in the kidney is not only correlated withinflammation pathology, but also that blocking chemokine action byanti-chemokine antibodies limits or halts progression of theinflammation and resulting tissue damage. The fact that normal humankidney expresses abundant NEOKINE transcript suggests that the kidneymakes significant quantities of the protein and that therefore thepro-inflammatory activity low or non-existent. This is consistent withthe proposed natural antagonist function of the NEOKINE proteins.Furthermore, signal peptide cleavage prediction on NEOKINE implies thatthe mature protein will have only two residues before the firstcysteine. In an analogous situation, an artificially truncated form ofIL-8 with only one residue before the first cysteine instead of thenaturally-occurring 6 residues converts the protein from an agonist toan antagonist.

Accordingly, in an exemplary embodiment, the modulatory method of theinvention involves contacting a cell with a NEOKINE such that theactivity of a chemokine is modulated. Alternatively, the modulatorymethod of the invention involves contacting a cell with a NEOKINE oragent that modulates one or more of the activities of NEOKINE proteinactivity associated with the cell. An agent that modulates NEOKINEprotein activity can be an agent as described herein, such as a nucleicacid or a protein, a naturally-occurring target molecule of a NEOKINEprotein (e.g., a carbohydrate), a NEOKINE antibody, a NEOKINE agonist orantagonist, a peptidomimetic of a NEOKINE agonist or antagonist, orother small molecule. In one embodiment, the agent stimulates one ormore NEOKINE activities. Examples of such stimulatory agents includeactive NEOKINE protein and a nucleic acid molecule encoding NEOKINE thathas been introduced into the cell. In another embodiment, the agentinhibits one or more NEOKINE activities. Examples of such inhibitoryagents include antisense NEOKINE nucleic acid molecules and anti-NEOKINEantibodies. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a NEOKINEprotein or nucleic acid molecule. Alternatively, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of achemokine. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) NEOKINE expression or activity or the expression oractivity of a chemokine. In another embodiment, the method involvesadministering a NEOKINE protein or nucleic acid molecule as therapy tocompensate for reduced or aberrant NEOKINE expression or activity. Inanother embodiment, the method involves administering a NEOKINE proteinor nucleic acid molecule as therapy to compensate for reduced oraberrant chemokine expression or activity.

A preferred embodiment of the present invention involves a method fortreatment of a SPOIL or NEOKINE associated disease or disorder whichincludes the step of administering a therapeutically effective amount ofa SPOIL or NEOKINE antibody to a subject. As defined herein, atherapeutically effective amount of antibody (i.e., an effective dosage)ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg bodyweight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisanwill appreciate that certain factors may influence the dosage requiredto effectively treat a subject, including but not limited to theseverity of the disease or disorder, previous treatments, the generalhealth and/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of anantibody can include a single treatment or, preferably, can include aseries of treatments. In a preferred example, a subject is treated withantibody in the range of between about 0.1 to 20 mg/kg body weight, onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody used for treatment may increase ordecrease over the course of a particular treatment. Changes in dosagemay result from the results of diagnostic assays as described herein.

Stimulation of FTHMA-070, Tango85, Tango77, Tango129 or A259 activity isdesirable in situations in which FTHMA-070, Tango85, Tango77, Tango129or A259 is abnormally downregulated and/or in which increased FTHMA-070,Tango85, Tango77, Tango129 or A259 activity is likely to have abeneficial effect. Conversely, inhibition of FTHMA-070, Tango85,Tango77, Tango129 or A259 activity is desirable in situations in whichFTHMA-070, Tango85, Tango77, Tango129 or A259 is abnormally upregulatedand/or in which decreased FTHMA-070, Tango85, Tango77, Tango129 or A259activity is likely to have a beneficial effect.

Stimulation of expression or activity is desirable in situations inwhich SPOIL and/or IL-1 is abnormally downregulated and/or in whichincreased expression or activity is likely to have a beneficial effect.Likewise, inhibition of expression or activity is desirable insituations in which SPOIL and/or IL-1 is abnormally upregulated and/orin which decreased expression or activity is likely to have a beneficialeffect. One example of such a situation is where a subject has adisorder characterized by aberrant cellular differentiation (e.g., abone resorption disorder). Another example of such a situation is wherethe subject has a proinflammatory disorder (e.g., sepsis) characterizedby an aberrant SPOIL and/or IL-1 response.

Stimulation of NEOKINE activity is desirable in situations in whichNEOKINE is abnormally downregulated and/or in which increased NEOKINEactivity is likely to have a beneficial effect. For example, stimulationof NEOKINE activity is desirable in situations in which a chemokine isupregulated and/or in which increased NEOKINE activity is likely to havea beneficial effect (e.g., a situation is where a subject has a disordercharacterized by aberrant angiogenesis or inflammation, such as kidneyinflammation. Likewise, inhibition of NEOKINE activity is desirable insituations in which NEOKINE is abnormally upregulated and/or in whichdecreased NEOKINE activity is likely to have a beneficial effect.

Delta3 Diagnostic and Prognostic Assays

The present methods provides means for determining if a subject is atrisk of developing a disorder characterized by an aberrant Delta3activity, such as aberrant cell proliferation, degeneration, and/ordifferentiation resulting for example in a neurodegenerative disease orcancer. The invention also provides methods for determining whether asubject is at risk of developing a disease or disorder associated withone or more specific alleles of a Delta3 gene. In fact, specific Delta3alleles may be associated with specific diseases or disorders. Forexample, at least one allele of hDelta3 is likely to be associated withthe neurological disease ACCPN. Accordingly, the invention providesmethods for determining whether a subject has or is at risk ofdeveloping a neurological disease, e.g., ACCPN. In another embodiment,the invention provides methods for determining whether a subject has oris at risk of developing a vascular disorder or a disorder associatedwith cell fate determination. In one embodiment, the invention comprisesdetermining the identity of the Delta3 allele in a subject and comparingthe molecular structure of the Delta3 gene of the subject with themolecular structure of a Delta3 gene from a subject which does not havethe neurological disease. Determining the molecular structure can be,e.g., determining the identity of at least one nucleotide, determiningthe nucleotide composition or determining the methylation pattern of thegene.

In one embodiment, the invention provides a method for determiningwhether a subject has genetic lesion in a Delta3 gene or a specificallelic variant of a polymorphic region in a Delta3 gene. The specificallele can be a mutant allele. In another embodiment, the inventionprovides methods for determining whether a subject has an aberrantDelta3 protein, resulting from aberrant post-translational modificationsof the protein, such as aberrant phosphorogulation or glycosylation.Also, within the scope of the invention are methods for determiningwhether a subject has an aberrant expression level of a Delta3 protein,which could be due to a genetic lesion in the Delta3 gene or due to anaberrant level or activity of a protein regulating the expression of aDelta3 gene.

In preferred embodiments, the methods can be characterized as comprisingdetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by at least one of (i) analteration affecting the integrity of a gene encoding a Delta-protein,or (ii) the mis-expression of a Delta3 gene. To illustrate, such geneticlesions can be detected by ascertaining the existence of at least one of(i) a deletion of one or more nucleotides from a Delta3 gene, (ii) anaddition of one or more nucleotides to a Delta3 gene, (iii) asubstitution of one or more nucleotides of a Delta3 gene, (iv) a grosschromosomal rearrangement of a Delta3 gene, (v) a gross alteration inthe level of a messenger RNA transcript of a Delta3 gene, (vii) aberrantmodification of a Delta3 gene, such as of the methylation pattern of thegenomic DNA, (vii) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a Delta3 gene, (viii) a non-wild type levelof a Delta-protein, (ix) allelic loss of a Delta3 gene, and (x)inappropriate post-translational modification of a Delta-protein. As setout below, the present invention provides a large number of assaytechniques for detecting lesions in a Delta3 gene, and importantly,provides the ability to discern between different molecular causesunderlying Delta-dependent aberrant cell proliferation and/ordifferentiation.

For determining whether a subject has or is at risk of developing adisease or condition associated with a specific allele of a Delta3 gene,preliminary experiments can be performed to determine the identity ofthe allele associated with a disease. For example, for determining theidentity of the hDelta3 allele associated with ACCPN, one can performmutation detection studies of the Delta3 gene in populations having ahigh risk of developing ACCPN. For example, one can perform mutationdetection analysis of the genomic DNA from subjects in the FrenchCanadian population in the Charlevoix and Saguenay-Lac St Jean regionsof the province of Quebec (Casaubon et al. (1996) Am. J. Hum. Genet.58:28). Such an analysis will reveal the identity of the Delta3 alleleor alleles associated with ACCPN. Comparison of the Delta3 allele of asubject with this allele or alleles associated with ACCPN will indicatewhether a subject has a Delta3 allele associated with ACCPN and thuswhether the subject has or is likely to develop ACCPN. Similarly,mutation detection analysis can also be carried out to determine theidentity of Delta3 alleles associated with other diseases or conditions.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a (purified) oligonucleotide probe including a region ofnucleotide sequence which is capable of hybridizing to a sense orantisense sequence of a Delta3 gene, such as represented by any of SEQID NOs: 1, 3, 24, 26, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45, allelesthereof, naturally-occurring mutants thereof, or 5′ or 3′ flankingsequences or intronic sequences naturally associated with the subjectDelta3 genes or naturally-occurring mutants thereof. The nucleic acid ofa cell is rendered accessible for hybridization, the probe is exposed tonucleic acid of the sample, and the hybridization of the probe to thesample nucleic acid is detected. Such techniques can be used to detectlesions at either the genomic or mRNA level, including deletions,substitutions, etc., as well as to determine mRNA transcript levels.

As set out above, one aspect of the present invention relates todiagnostic assays for determining, in the context of cells isolated froma patient, if mutations have arisen in one or more Delta3 genes of thesample cells. The present method provides a method for determining if asubject is at risk for a disorder characterized by aberrant Delta3activity, e.g., cell proliferation and/or differentiation. In preferredembodiments, the method can be generally characterized as comprisingdetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion characterized by an alteration affecting theintegrity of a gene encoding a Delta protein. To illustrate, suchgenetic lesions can be detected by ascertaining the existence of atleast one of (i) a deletion of one or more nucleotides from aDelta-gene, (ii) an addition of one or more nucleotides to a Delta-gene,(iii) a substitution of one or more nucleotides of a Delta-gene, and(iv) the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a Delta-gene. As set out below, the present inventionprovides a large number of assay techniques for detecting lesions inDelta3 genes, and importantly, provides the ability to discern betweendifferent molecular causes underlying Delta-dependent aberrant cellproliferation and/or differentiation.

In certain embodiments, detection of the lesion in a Delta gene or theidentity of an allelic variant of a polymorphic region of a Delta genecomprises utilizing the probe/primer in a polymerase chain reaction(PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such asanchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; andNakazawa et al. (1994) PNAS 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the Delta-gene (seeAbravaya et al. (1995) Nuc Acid Res 23:675-682). In a merelyillustrative embodiment, the method includes the steps of (i) collectinga sample of cells from a patient, (ii) isolating nucleic acid (e.g.,genomic, mRNA or both) from the cells of the sample, (iii) contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a Delta gene under conditions such that hybridization andamplification of the Delta-gene (if present) occurs, and (iv) detectingthe presence or absence of an amplification product, or detecting thesize of the amplification product and comparing the length to a controlsample. It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In a preferred embodiment of the subject assay, mutations in a Delta3gene or specific alleles of a Delta3 gene from a sample cell areidentified by alterations in restriction enzyme cleavage patterns. Forexample, sample and control DNA is isolated, amplified (optionally),digested with one or more restriction endonucleases, and fragment lengthsizes are determined by gel electrophoresis. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531,incorporated herein by reference in its entirety) can be used to scorefor the presence of specific mutations by development or loss of aribozyme cleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the Delta3 gene anddetect mutations or allelic variants of polymorphic regions by comparingthe sequence of the sample Delta3 with the corresponding wild-type(control) sequence. Exemplary sequencing reactions include those basedon techniques developed by Maxim and Gilbert (Proc. Natl. Acad Sci USA(1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci.74:5463). It is also contemplated that any of a variety of automatedsequencing procedures may be utilized when performing the subject assays(Biotechniques (1995) 19:448), including by sequencing by massspectrometry (see, for example PCT publication WO 94/16101; Cohen et al.(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159). It will be evident to one skilled in the artthat, for certain embodiments, the occurrence of only one, two or threeof the nucleic acid bases need be determined in the sequencing reaction.For instance, A-tract or the like, e.g., where only one nucleic acid isdetected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes(Myers, et al. (1985) Science 230:1242). In general, the art techniqueof “mismatch cleavage” starts by providing heteroduplexes of formed byhybridizing (labeled) RNA or DNA containing the wild-type Delta3sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to basepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl. Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymod. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in Delta3 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a Delta3sequence, e.g., a wild-type Delta3 sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039, incorporated herein by reference in itsentirety.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in Delta3 genes or for determining theidentity of the Delta3 allele. For example, single strand conformationpolymorphism (SSCP) may be used to detect differences in electrophoreticmobility between mutant and wild type nucleic acids (Orita et al. (1989)Proc Natl. Acad. Sci. USA 86:2766, see also Cotton (1993) Mutat Res285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).Single-stranded DNA fragments of sample and control Delta3 nucleic acidswill be denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labelled ordetected with labelled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotide hybridizationtechniques may be used to test one mutation per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations when the oligonucleotides are attached to thehybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238. In additionit may be desirable to introduce a novel restriction site in the regionof the mutation to create cleavage-based detection (Gasparini et al.(1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

Another embodiment of the invention provides for a nucleic acidcomposition comprising a (purified) oligonucleotide probe including aregion of nucleotide sequence which is capable of hybridizing to a senseor antisense sequence of a Delta-gene, or naturally-occurring mutantsthereof, or 5′ or 3′ flanking sequences or intronic sequences naturallyassociated with the subject Delta-genes or naturally-occurring mutantsthereof. The nucleic acid of a cell is rendered accessible forhybridization, the probe is exposed to nucleic acid of the sample, andthe hybridization of the probe to the sample nucleic acid is detected.Such techniques can be used to detect lesions at either the genomic ormRNA level, including deletions, substitutions, etc., as well as todetermine mRNA transcript levels. Such oligonucleotide probes can beused for both predictive and therapeutic evaluation of allelic mutationswhich might be manifest in, for example, a neurodegenerative, neoplasticor hyperplastic disorders (e.g., aberrant cell growth).

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a Delta3 gene.

Any cell type or tissue, preferably neural or endothelial cells, inwhich the Delta3 is expressed may be utilized in the diagnosticsdescribed below. For example, a subject's bodily fluid (e.g., blood) canbe obtained by known techniques (e.g., venipuncture). Alternatively,nucleic acid tests can be performed on dry samples (e.g., hair or skin).Fetal nucleic acid samples can be obtained from maternal blood asdescribed in International Patent Application NO: WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing, e.g., of ACCPN, which is a disease which isusually fatal in the third decade of life.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Antibodies directed against wild type or mutant Delta3 proteins, whichare discussed, above, may also be used in disease diagnostics andprognostics. Such diagnostic methods, may be used to detectabnormalities in the level of Delta3 protein expression, orabnormalities in the structure and/or tissue, cellular, or subcellularlocation of Delta3 proteins. Structural differences may include, forexample, differences in the size, electronegativity, or antigenicity ofthe mutant Delta3 protein relative to the normal Delta3 protein. Proteinfrom the tissue or cell type to be analyzed may easily be detected orisolated using techniques which are well known to one of skill in theart, including but not limited to western blot analysis. For a detailedexplanation of methods for carrying out western blot analysis, seeSambrook et al, 1989, supra, at Chapter 18. The protein detection andisolation methods employed herein may also be such as those described inHarlow and Lane, for example, (Harlow, E. and Lane, D., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of Delta3 proteins. Insitu detection may be accomplished by removing a histological specimenfrom a patient, and applying thereto a labeled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the Delta3 protein, but also its distribution inthe examined tissue. Using the present invention, one of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an anti-Delta3 protein specific antibody is vialinkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller,“The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978);Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986). The radioactive isotope can be detected by such means asthe use of a gamma counter or a scintillation counter or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods fordetecting alterations in a Delta3 gene or gene product can be used tomonitor the course of treatment or therapy.

Delta3 Drug Screening Assays

The invention provides for compounds, e.g., therapeutic compounds, fortreating diseases or conditions caused by, or contributed to by anabnormal Delta3 activity. The compounds that can be used for thispurpose can be any type of compound, including a protein, a peptide,peptidomimetic, small molecule, and nucleic acid. A nucleic acid can be,e.g., a gene, an antisense nucleic acid, a ribozyme, or a triplexmolecule. A compound of the invention can be an agonist or anantagonist. A compound can act on a Delta3 gene, e.g., to modulate itsexpression. A compound can also act on a Delta3 protein, e.g., tomodulate signal transduction from the receptor. Accordingly, a compoundof the invention can be a compound which binds to Delta3 and inducessignal transduction from the receptor, such that, e.g., a Delta3activity is induced. Alternatively, a compound of the invention can be acompound which inhibits interaction of a Delta3 protein with atoporythmic protein, e.g., Notch. In one embodiment, a compound of theinvention which interacts with a Delta protein, which is either anagonist or an antagonist, is a toporythmic protein or other proteininteracting with Delta3. In an even more preferred embodiment, thecompound is a soluble toporythmic protein or other protein interactingwith Delta3. For example, a soluble antagonistic toporythmic protein canbe a protein which competes with the wild type toporythmic proteins forbinding to Delta3. A soluble agonistic toporythmic protein can be aprotein which binds to a Delta3 protein in essentially the same manneras a wild-type toporythmic protein, such as to induce at least oneDelta3 activity, e.g., signal transduction from the Delta3 protein.Accordingly, a soluble toporythmic protein can be stimulatory form of atoporythmic protein or an inhibitory form of a toporythmic, depending onwhether the particular toporythmic protein stimulates or inhibits aDelta3 activity.

Similarly, a soluble Delta3 protein, e.g., Delta3-Ig, can be used tomodulate an activity of a toporythmic protein, e.g., Notch. For example,a soluble Delta3 protein can be a stimulatory form of a Delta3 protein,i.e., a Delta3 protein which is capable of stimulating an activity of atoporythmic protein. In one embodiment, such a protein acts inessentially the same manner as wild-type Delta3. In another embodiment,a soluble Delta3 protein is an inhibitory form of a Delta3 protein,i.e., a Delta3 protein which is capable of inhibiting an activity of atoporythmic protein. For example, such a Delta3 protein could inhibitthe interaction of wild-type Delta3 with the toporythmic protein. In apreferred embodiment, an inhibitory form of a Delta3 protein inhibitsthe interaction of several proteins which normally interact with atoporythmic protein, by, e.g., binding to a site of the toporythmicprotein that is also a binding site to various other proteins, e.g.,other Delta proteins. Accordingly, a Delta3 therapeutic can generallyaffect the interaction of various toporythmic proteins with each other.Similarly, based at least in part on the sequence and structuralsimilarities between Delta proteins, a Delta therapeutic, other than aDelta3 therapeutic, can also be used for modulating the interactionbetween a Delta3 protein and a Delta3 interacting binding molecule.

The compounds of the invention can be identified using various assaysdepending on the type of compound and activity of the compound that isdesired. Set forth below are at least some assays that can be used foridentifying Delta3 therapeutics. It is within the skill of the art todesign additional assays for identifying Delta therapeutics, e.g.,Delta3 therapeutics.

By making available purified and recombinant Delta3 polypeptides, thepresent invention facilitates the development of assays which can beused to screen for drugs, including Delta3 variants, which are eitheragonists or antagonists of the normal cellular function of the subjectDelta3 polypeptides, or of their role in the pathogenesis of cellulardifferentiation and/or proliferation and disorders related thereto. Inone embodiment, the assay evaluates the ability of a compound tomodulate binding between a Delta3 polypeptide and a molecule, be itprotein or DNA, that interacts either upstream or downstream of theDelta/Notch signaling pathway. A variety of assay formats will sufficeand, in light of the present inventions, will be comprehended by askilled artisan.

Delta3 Cell-Free Assays

Cell free assays can be used to identify compounds which interact with aDelta3 protein. Such assays are available for testing compounds whichare proteins, e.g., toporythmic proteins or variants thereof, as well asfor testing compounds which are peptidomimetics, small molecules ornucleic acids. The specific assay used for testing these compounds mayvary with the type of compound.

In one embodiment, a compound that interacts with a Delta3 protein isidentified by screening, e.g., a library of compounds, for binding to arecombinant or purified Delta3 protein or at least a portion thereof.Such assays can involve labeling one or the two components and measuringthe extent of their interaction, by, e.g., determining the level of theone or two labels. In these assays, it may be preferable to attach theDelta3 protein to a solid phase surface. Methods for achieving this arefurther described infra. In one embodiment, the library of compounds isa library of small molecules. In another embodiment, the library ofcompounds is a library of Delta3 variants, which can be producedaccording to methods described infra.

Identification of a compound which inhibits an interaction between aDelta3 protein and a toporythmic protein can also be performed byscreening compounds using aggregation assays, as described, e.g., inFehon et al. (1990) Cell 61:523-534.

In another embodiment, the invention provides methods for identifyingcompounds which inhibit the interaction of a Delta3 protein with amolecule, e.g., a toporythmic protein or a protein interacting with thecytoplasmic domain of a Delta3 protein. Such methods, which arepreferably used in high throughput assays can be performed as follows.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, the compound of interest is contacted with proteinswhich may function upstream (including both activators and repressors ofits activity) or to proteins or nucleic acids which may functiondownstream of the Delta3 polypeptide, whether they are positively ornegatively regulated by it. For example, a protein functioning upstreamof a Delta3 polypeptide can be a compound interacting with theextracellular portion of the Delta3 molecule. A protein functioningdownstream of a Delta3 polypeptide can be a protein interacting with thecytoplasmic domain of Delta3 and, e.g., transducing a signal to thenucleus. To the mixture of the compound and the upstream or downstreamelement is then added a composition containing a Delta3 polypeptide.Detection and quantification of complexes of Delta3 with it's upstreamor downstream elements provide a means for determining a compound'sefficacy at inhibiting (or potentiating) complex formation betweenDelta3 and the Delta-binding elements. The efficacy of the compound canbe assessed by generating dose response curves from data obtained usingvarious concentrations of the test compound. Moreover, a control assaycan also be performed to provide a baseline for comparison. In thecontrol assay, isolated and purified Delta3 polypeptide is added to acomposition containing the Delta-binding element, and the formation of acomplex is quantitated in the absence of the test compound.

Complex formation between the Delta3 polypeptide and a Delta3 bindingelement may be detected by a variety of techniques. Modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled, fluorescently labeled, orenzymatically labeled Delta3 polypeptides, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either Delta3 or itsbinding protein to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of Delta3 to an upstream or downstreamelement, in the presence and absence of a candidate agent, can beaccomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/Delta3 (GST/Delta) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates, e.g., an ³⁵S-labeled, and the testcompound, and the mixture incubated under conditions conducive tocomplex formation, e.g., at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g., beadsplaced in scintilant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofDelta-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques such as described in theappended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either Delta3 orits cognate binding protein can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated Delta3 molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withDelta3 but which do not interfere with binding of upstream or downstreamelements can be derivatized to the wells of the plate, and Delta3trapped in the wells by antibody conjugation. As above, preparations ofa Delta-binding protein and a test compound are incubated in theDelta-presenting wells of the plate, and the amount of complex trappedin the well can be quantitated. Exemplary methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the Delta3 binding element, or which are reactive withDelta3 protein and compete with the binding element; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the binding element, either intrinsic or extrinsicactivity. In the instance of the latter, the enzyme can be chemicallyconjugated or provided as a fusion protein with the Delta-BP. Toillustrate, the Delta-BP can be chemically cross-linked or geneticallyfused with horseradish peroxidase, and the amount of polypeptide trappedin the complex can be assessed with a chromogenic substrate of theenzyme, e.g., 3,3′-diamino-benzadine terahydrochloride or4-chloro-1-naphthol. Likewise, a fusion protein comprising thepolypeptide and glutathione-S-transferase can be provided, and complexformation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-Delta3 antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the Delta3 sequence, a secondpolypeptide for which antibodies are readily available (e.g., fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharmacia, NJ).

Delta3 Cell Based Assays

In addition to cell-free assays, such as described above, the readilyavailable source of Delta3 proteins provided by the present inventionalso facilitates the generation of cell-based assays for identifyingsmall molecule agonists/antagonists and the like. For example, cellswhich are sensitive to bFGF/VEGF or matrigel can be caused tooverexpress a recombinant Delta3 protein in the presence and absence ofa test agent of interest, with the assay scoring for modulation inDelta3 responses by the target cell mediated by the test agent. As withthe cell-free assays, agents which produce a statistically significantchange in Delta-dependent responses (either inhibition or potentiation)can be identified. In an illustrative embodiment, the expression oractivity of a Delta3 is modulated in embryos or cells and the effects ofcompounds of interest on the readout of interest (such as tissuedifferentiation, proliferation, tumorigenesis) are measured. Forexample, the expression of genes which are up- or down-regulated inresponse to a Delta-dependent signal cascade can be assayed. Inpreferred embodiments, the regulatory regions of such genes, e.g., the5′ flanking promoter and enhancer regions, are operably linked to adetectable marker (such as luciferase) which encodes a gene product thatcan be readily detected.

Exemplary cell lines may include endothelial cells such as MVEC's andbovine aortic endothelial cells (BAEC's); as well as generic mammaliancell lines such as HeLa cells and COS cells, e.g., COS-7 (ATCC®#CRL-1651). Further, the transgenic animals discussed herein may be usedto generate cell lines, containing one or more cell types involved incardiovascular disease, that can be used as cell culture models for thisdisorder. While primary cultures derived from the transgenic animals ofthe invention may be utilized, the generation of continuous cell linesis preferred. For examples of techniques which may be used to derive acontinuous cell line from the transgenic animals, see Small et al.,1985, Mol. Cell. Biol. 5:642-648.

In one embodiment, a test compound that modifies a Delta3 activity canbe identified by incubating a cell having a Delta3 protein with the testcompound and measuring signal transduction from the Delta3 protein.Comparison of the signal transduction in the cells incubated with orwithout the test compound will reveal whether the test compound is aDelta3 therapeutic. Similarly, a test compound that modifies a Delta3activity can be identified by incubating a cell having a Delta3 ligandwith the test compound, e.g., a Delta3 derived compound, and measuringsignal transduction from the Delta3 ligand. Comparison of the signaltransduction in the cells incubated with or without the test compoundwill reveal whether the test compound is a Delta3 therapeutic.

In the event that the Delta3 proteins themselves, or in complexes withother proteins, are capable of binding DNA and/or modifyingtranscription of a gene, a transcriptional based assay could be used,for example, in which a Delta3 responsive regulatory sequence isoperably linked to a detectable marker gene, e.g., a luciferase gene.Similarly, Delta3 therapeutics could also be identified by using anassay in which expression of genes that are modulated upon binding of aDelta3 protein to a Delta3 ligand on a cell is monitored. Genes that areresponsive to interaction with a Delta3 protein or Delta3 ligand can beidentified according to methods known in the art, e.g., differentialhybridization or differential display.

In another embodiment, a silicon-based device, called amicrophysiometer, can be used to detect and measure the response ofcells having a Delta3 protein to test compounds to identify Delta3therapeutics. This instrument measures the rate at which cells acidifytheir environment, which is indicative of cellular growth and/ordifferentiation (McConnel et al. (1992) Science 257:1906).

Monitoring the influence of compounds on cells may be applied not onlyin basic drug screening, but also in clinical trials. In such clinicaltrials, the expression of a panel of genes may be used as a “read out”of a particular drug's therapeutic effect.

In yet another aspect of the invention, the subject Delta3 polypeptidescan be used to generate a “two hybrid” assay (see, for example, U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), for isolating coding sequences for other cellular proteinswhich bind to or interact with Delta3 (“Delta-binding proteins” or“Delta-bp”), such as Notch, and the like.

Briefly, the two hybrid assay relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for a Delta3polypeptide. The second hybrid protein encodes a transcriptionalactivation domain fused in frame to a sample gene from a cDNA library.If the bait and sample hybrid proteins are able to interact, e.g., forma Delta-dependent complex, they bring into close proximity the twodomains of the transcriptional activator. This proximity is sufficientto cause transcription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the reporter gene can be detected and usedto score for the interaction of the Delta3 and sample proteins. Thissystem can be used to identify compounds which modify, e.g., inhibit theinteraction between a Delta3 protein and another protein, by adding 1test compound to a cell containing the above-described plasmids. Theeffect of the test compound on the reporter gene expression and thenmeasured to determine the effect of the test compound on theinteraction.

In another embodiment, the invention provides arrays for identifyingcompounds that can induce apoptosis of cells through a Delta3 protein.Apoptotic arrays are known in the act and are described, e.g., in Grimmet al. (1996) Proc. Natl. Acad. Sci. USA 93:10923.

Delta3 Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

Delta3 Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, nucleic acid molecules described herein orfragments thereof, can be used to map the location of the correspondinggenes on a chromosome. The mapping of the sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, genes can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp in length) from the sequence of a gene of theinvention. Computer analysis of the sequence of a gene of the inventioncan be used to rapidly select primers that do not span more than oneexon in the genomic DNA, thus complicating the amplification process.These primers can then be used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the gene sequences will yield anamplified fragment. For a review of this technique, see D'Eustachio etal. ((1983) Science 220:919-924).

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the nucleicacid sequences of the invention to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa gene to its chromosome include in situ hybridization (described in Fanet al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening withlabeled flow-sorted chromosomes (CITE), and pre-selection byhybridization to chromosome specific cDNA libraries. Fluorescence insitu hybridization (FISH) of a DNA sequence to a metaphase chromosomalspread can further be used to provide a precise chromosomal location inone step. For a review of this technique, see Verma et al., (HumanChromosomes: A Manual of Basic Techniques (Pergamon Press, New York,1988)).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with a gene of the inventioncan be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

Furthermore, the nucleic acid sequences disclosed herein can be used toperform searches against “mapping databases”, e.g., BLAST-type search,such that the chromosome position of the gene is identified by sequencehomology or identity with known sequence fragments which have beenmapped to chromosomes.

A polypeptide and fragments and sequences thereof and antibodiesspecific thereto can be used to map the location of the gene encodingthe polypeptide on a chromosome. This mapping can be carried out byspecifically detecting the presence of the polypeptide in members of apanel of somatic cell hybrids between cells of a first species of animalfrom which the protein originates and cells from a second species ofanimal and then determining which somatic cell hybrid(s) expresses thepolypeptide and noting the chromosome(s) from the first species ofanimal that it contains. For examples of this technique, see Pajunen etal. (1988) Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al. (1986)Hum. Genet. 74:34-40. Alternatively, the presence of the polypeptide inthe somatic cell hybrids can be determined by assaying an activity orproperty of the polypeptide, for example, enzymatic activity, asdescribed in Bordelon-Riser et al. (1979) Somatic Cell Genetics5:597-613 and Owerbach et al. (1978) Proc. Natl. Acad. Sci. USA75:5640-5644.

Delta3 Tissue Typing

The nucleic acid sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the nucleic acid sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The nucleic acid sequences of the invention uniquely represent portionsof the human genome. Allelic variation occurs to some degree in thecoding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO: 1 and24, can comfortably provide positive individual identification with apanel of perhaps 10 to 1,000 primers which each yield a noncodingamplified sequence of 100 bases. If predicted coding sequences, such asthose in SEQ ID NO: 3 and 25 are used, a more appropriate number ofprimers for positive individual identification would be 500-2,000.

If a panel of reagents from the nucleic acid sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

Delta3 Use of Partial Gene Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the nucleic acid sequencesof the invention or portions thereof, e.g., fragments derived fromnoncoding regions having a length of at least 20 or 30 bases.

The nucleic acid sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such probes can be used to identify tissue byspecies and/or by organ type.

Delta3 Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningexpression of a polypeptide or nucleic acid of the invention and/oractivity of a polypeptide of the invention, in the context of abiological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrantexpression or activity of a polypeptide of the invention. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith aberrant expression or activity of a polypeptide of the invention.For example, mutations in a gene of the invention can be assayed in abiological sample. Such assays can be used for prognostic or predictivepurpose to thereby prophylactically treat an individual prior to theonset of a disorder characterized by or associated with aberrantexpression or activity of a polypeptide of the invention.

Another aspect of the invention provides methods for expression of anucleic acid or polypeptide of the invention or activity of apolypeptide of the invention in an individual to thereby selectappropriate therapeutic or prophylactic agents for that individual(referred to herein as “pharmacogenomics”). Pharmacogenomics allows forthe selection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the genotype of the individual(e.g., the genotype of the individual examined to determine the abilityof the individual to respond to a particular agent).

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof a polypeptide of the invention in clinical trials. These and otheragents are described in further detail in the following sections.

Delta3 Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of a polypeptide of the invention. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with aberrant expression oractivity of a polypeptide of the invention. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing such a disease or disorder. Thus, the presentinvention provides a method in which a test sample is obtained from asubject and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) ofthe invention is detected, wherein the presence of the polypeptide ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant expression or activity ofthe polypeptide. As used herein, a “test sample” refers to a biologicalsample obtained from a subject of interest. For example, a test samplecan be a biological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant expression or activity of a polypeptide of theinvention. For example, such methods can be used to determine whether asubject can be effectively treated with a specific agent or class ofagents (e.g., agents of a type which decrease activity of thepolypeptide). Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant expression or activity of apolypeptide of the invention in which a test sample is obtained and thepolypeptide or nucleic acid encoding the polypeptide is detected (e.g.,wherein the presence of the polypeptide or nucleic acid is diagnosticfor a subject that can be administered the agent to treat a disorderassociated with aberrant expression or activity of the polypeptide).

The methods of the invention can also be used to detect genetic lesionsor mutations in a gene of the invention, thereby determining if asubject with the lesioned gene is at risk for a disorder characterizedaberrant expression or activity of a polypeptide of the invention. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesion ormutation characterized by at least one of an alteration affecting theintegrity of a gene encoding the polypeptide of the invention, or themis-expression of the gene encoding the polypeptide of the invention.For example, such genetic lesions or mutations can be detected byascertaining the existence of at least one of: 1) a deletion of one ormore nucleotides from the gene; 2) an addition of one or morenucleotides to the gene; 3) a substitution of one or more nucleotides ofthe gene; 4) a chromosomal rearrangement of the gene; 5) an alterationin the level of a messenger RNA transcript of the gene; 6) an aberrantmodification of the gene, such as of the methylation pattern of thegenomic DNA; 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of the gene; 8) a non-wild type level of a theprotein encoded by the gene; 9) an allelic loss of the gene; and 10) aninappropriate post-translational modification of the protein encoded bythe gene. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in agene.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a gene (see, e.g.,Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to the selected gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a selected gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations can be identified by hybridizinga sample and control nucleic acids, e.g., DNA or RNA, to high densityarrays containing hundreds or thousands of oligonucleotides probes(Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996)Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin et al. (1996) Human Mutation 7:244-255.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the selected gene anddetect mutations by comparing the sequence of the sample nucleic acidswith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNO: WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in a selected gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the technique of mismatch cleavage entailsproviding heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type sequence with potentially mutant RNA or DNAobtained from a tissue sample. The double-stranded duplexes are treatedwith an agent which cleaves single-stranded regions of the duplex suchas which will exist due to basepair mismatches between the control andsample strands. RNA/DNA duplexes can be treated with RNase to digestmismatched regions, and DNA/DNA hybrids can be treated with S1 nucleaseto digest mismatched regions.

In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g., Cottonet al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair enzymes”) in defined systems fordetecting and mapping point mutations in cDNAs obtained from samples ofcells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a selectedsequence, e.g., a wild-type sequence, is hybridized to a cDNA or otherDNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, e.g., U.S.Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). Single-stranded DNA fragments of sample and control nucleicacids will be denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, and theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient get electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a gene encoding apolypeptide of the invention. Furthermore, any cell type or tissue,preferably peripheral blood leukocytes, in which the polypeptide of theinvention is expressed may be utilized in the prognostic assaysdescribed herein.

Delta3 Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onactivity or expression of a polypeptide of the invention as identifiedby a screening assay described herein can be administered to individualsto treat (prophylactically or therapeutically) disorders associated withaberrant activity of the polypeptide. In conjunction with suchtreatment, the pharmacogenomics (i.e., the study of the relationshipbetween an individual's genotype and that individual's response to aforeign compound or drug) of the individual may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of a polypeptide of the invention,expression of a nucleic acid of the invention, or mutation content of agene of the invention in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of a polypeptide of the invention, expression of anucleic acid encoding the polypeptide, or mutation content of a geneencoding the polypeptide in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used toapply genotyping of polymorphic alleles encoding drug-metabolizingenzymes to the identification of an individual's drug responsivenessphenotype. This knowledge, when applied to dosing or drug selection, canavoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with amodulator of activity or expression of the polypeptide, such as amodulator identified by one of the exemplary screening assays describedherein.

Delta3 Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of a polypeptide of the invention (e.g., theability to modulate aberrant cell proliferation chemotaxis, and/ordifferentiation) can be applied not only in basic drug screening, butalso in clinical trials. For example, the effectiveness of an agent, asdetermined by a screening assay as described herein, to increase geneexpression, protein levels or protein activity, can be monitored inclinical trials of subjects exhibiting decreased gene expression,protein levels, or protein activity. Alternatively, the effectiveness ofan agent, as determined by a screening assay, to decrease geneexpression, protein levels or protein activity, can be monitored inclinical trials of subjects exhibiting increased gene expression,protein levels, or protein activity. In such clinical trials, expressionor activity of a polypeptide of the invention and preferably, that ofother polypeptide that have been implicated in for example, a cellularproliferation disorder, can be used as a marker of the immuneresponsiveness of a particular cell.

For example, and not by way of limitation, genes, including those of theinvention, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates activity or expressionof a polypeptide of the invention (e.g., as identified in a screeningassay described herein) can be identified. Thus, to study the effect ofagents on cellular proliferation disorders, for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of a gene of the invention and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of a gene of the invention or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of the polypeptide or nucleic acidof the invention in the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thelevel the of the polypeptide or nucleic acid of the invention in thepost-administration samples; (v) comparing the level of the polypeptideor nucleic acid of the invention in the pre-administration sample withthe level of the polypeptide or nucleic acid of the invention in thepost-administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of the polypeptide to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of the polypeptide to lower levels thandetected, i.e., to decrease the effectiveness of the agent.

Delta3 Transgenic Animals

These systems may be used in a variety of applications. For example, thecell- and animal-based model systems may be used to further characterizeDelta3 genes and proteins. In addition, such assays may be utilized aspart of screening strategies designed to identify compounds which arecapable of ameliorating disease symptoms. Thus, the animal- andcell-based models may be used to identify drugs, pharmaceuticals,therapies and interventions which may be effective in treating disease.

Delta3 Animal-Based Systems

One aspect of the present invention concerns transgenic animals whichare comprised of cells (of that animal) which contain a transgene of thepresent invention and which preferably (though optionally) express anexogenous Delta3 protein in one or more cells in the animal. A Delta3transgene can encode the wild-type form of the protein, or can encodehomologs thereof, including both alleles of Delta3 genes, agonists andantagonists, as well as antisense constructs. In preferred embodiments,the expression of the transgene is restricted to specific subsets ofcells, tissues or developmental stages utilizing, for example,cis-acting sequences that control expression in the desired pattern. Inthe present invention, such mosaic expression of a Delta3 protein can beessential for many forms of lineage analysis and can additionallyprovide a means to assess the effects of, for example, lack of Delta3expression which might grossly alter development in small patches oftissue within an otherwise normal embryo. In a preferred embodiment, theinvention provides transgenic mice having an allele of hDelta3 genewhich is associated with ACCPN and the mouse can be used, e.g., todetermine the effect of this specific hDelta3 allele. Toward this and,tissue-specific regulatory sequences and conditional regulatorysequences can be used to control expression of the transgene in certainspatial patterns. Moreover, temporal patterns of expression can beprovided by, for example, conditional recombination systems orprokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject Delta3 proteins. For example, excisionof a target sequence which interferes with the expression of arecombinant Delta3 gene, such as one which encodes an antagonistichomolog or an antisense transcript, can be designed to activateexpression of that gene. This interference with expression of theprotein can result from a variety of mechanisms, such as spatialseparation of the Delta3 gene from the promoter element or an internalstop codon. Moreover, the transgene can be made wherein the codingsequence of the gene is flanked by recombinase recognition sequences andis initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

The transgenic animals of the present invention all include within aplurality of their cells a transgene of the present invention, whichtransgene alters the phenotype of the “host cell” with respect toregulation of cell growth, death and/or differentiation. Since it ispossible to produce transgenic organisms of the invention utilizing oneor more of the transgene constructs described herein, a generaldescription will be given of the production of transgenic organisms byreferring generally to exogenous genetic material. This generaldescription can be adapted by those skilled in the art in order toincorporate specific transgene sequences into organisms utilizing themethods and materials described below.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotiderepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a recombinant Delta3 protein can beregulated via control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant Delta3 protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant Delta3 gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., a Delta3 gene and recombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a Delta3 transgene in a recombinase-mediated expressibleformat derives from the likelihood that the subject protein, whetheragonistic or antagonistic, can be deleterious upon expression in thetransgenic animal. In such an instance, a founder population, in whichthe subject transgene is silent in all tissues, can be propagated andmaintained. Individuals of this founder population can be crossed withanimals expressing the recombinase in, for example, one or more tissuesand/or a desired temporal pattern. Thus, the creation of a founderpopulation in which, for example, an antagonistic Delta3 transgene issilent will allow the study of progeny from that founder in whichdisruption of Delta3 mediated induction in a particular tissue or atcertain developmental stages would result in, for example, a lethalphenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the Delta3 transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the trans-activatingprotein, e.g., a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, a Delta3 transgene could remain silent intoadulthood until “turned on” by the introduction of the trans-activator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/1. The line(s) usedto practice this invention may themselves be transgenics, and/or may beknockouts (i.e., obtained from animals which have one or more genespartially or completely suppressed).

In one embodiment, the transgene construct is introduced into a singlestage embryo. The zygote is the best target for micro-injection. In themouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 μl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the nucleotide sequence comprisingthe transgene is introduced into the female or male pronucleus asdescribed below. In some species such as mice, the male pronucleus ispreferred. It is most preferred that the exogenous genetic material beadded to the male DNA complement of the zygote prior to its beingprocessed by the ovum nucleus or the zygote female pronucleus. It isthought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

Thus, it is preferred that the exogenous genetic material be added tothe male complement of DNA or any other complement of DNA prior to itsbeing affected by the female pronucleus. For example, the exogenousgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcan then be added to the ovum or the decondensed sperm could be added tothe ovum with the transgene constructs being added as soon as possiblethereafter.

Introduction of the transgene nucleotide sequence into the embryo may beaccomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, i.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA sequenceswill vary depending upon the particular zygote and functions of theexogenous genetic material and will be readily apparent to one skilledin the art, because the genetic material, including the exogenousgenetic material, of the resulting zygote must be biologically capableof initiating and maintaining the differentiation and development of thezygote into a functional organism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional. Asregards the present invention, there will often be an advantage tohaving more than one functioning copy of each of the inserted exogenousDNA sequences to enhance the phenotypic expression of the exogenous DNAsequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the present inventionwill include exogenous genetic material. As set out above, the exogenousgenetic material will, in certain embodiments, be a DNA sequence whichresults in the production of a Delta3 protein (either agonistic orantagonistic), and antisense transcript, or a Delta3 mutant. Further, insuch embodiments the sequence will be attached to a transcriptionalcontrol element, e.g., a promoter, which preferably allows theexpression of the transgene product in a specific type of cell.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten et al. (1985) PNAS 82:6148-6152; Stewart et al. (1987) EMBOJ. 6:383-388). Alternatively, infection can be performed at a laterstage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al. (1982) Nature 298:623-628). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic non-humananimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line by intrauterine retroviralinfection of the midgestation embryo (Jahner et al. (1982) Nature298:623-628).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

In one embodiment, gene targeting, which is a method of using homologousrecombination to modify an animal's genome, can be used to introducechanges into cultured embryonic stem cells. By targeting a Delta3 geneof interest in ES cells, these changes can be introduced into thegermlines of animals to generate chimeras. The gene targeting procedureis accomplished by introducing into tissue culture cells a DNA targetingconstruct that includes a segment homologous to a target Delta3 locus,and which also includes an intended sequence modification to the Delta3genomic sequence (e.g., insertion, deletion, point mutation). Thetreated cells are then screened for accurate targeting to identify andisolate those which have been properly targeted.

Gene targeting in embryonic stem cells is in fact a scheme contemplatedby the present invention as a means for disrupting a Delta3 genefunction through the use of a targeting transgene construct designed toundergo homologous recombination with one or more Delta3 genomicsequences. The targeting construct can be arranged so that, uponrecombination with an element of a Delta3 gene, a positive selectionmarker is inserted into (or replaces) coding sequences of the targetedDelta3 gene. The inserted sequence functionally disrupts the Delta3gene, while also providing a positive selection trait. Exemplary Delta3targeting constructs are described in more detail below.

Generally, the embryonic stem cells (ES cells) used to produce theknockout animals will be of the same species as the knockout animal tobe generated. Thus for example, mouse embryonic stem cells will usuallybe used for generation of knockout mice.

Embryonic stem cells are generated and maintained using methods wellknown to the skilled artisan such as those described by Doetschman etal. (1985) J. Embryol. Exp. Morphol. 87:27-45). Any line of ES cells canbe used, however, the line chosen is typically selected for the abilityof the cells to integrate into and become part of the germ line of adeveloping embryo so as to create germ line transmission of the knockoutconstruct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog NO: CKL 1934) Still another preferred ES cell line is the WW6cell line (Ioffe et al. (1995) PNAS 92:7357-7361). The cells arecultured and prepared for knockout construct insertion using methodswell known to the skilled artisan, such as those set forth by Robertsonin: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1986]).

Insertion of the knockout construct into the ES cells can beaccomplished using a variety of methods well known in the art includingfor example, electroporation, microinjection, and calcium phosphatetreatment. A preferred method of insertion is electroporation.

Each knockout construct to be inserted into the cell must first be inthe linear form. Therefore, if the knockout construct has been insertedinto a vector (described infra), linearization is accomplished bydigesting the DNA with a suitable restriction endonuclease selected tocut only within the vector sequence and not within the knockoutconstruct sequence.

For insertion, the knockout construct is added to the ES cells underappropriate conditions for the insertion method chosen, as is known tothe skilled artisan. Where more than one construct is to be introducedinto the ES cell, each knockout construct can be introducedsimultaneously or one at a time.

If the ES cells are to be electroporated, the ES cells and knockoutconstruct DNA are exposed to an electric pulse using an electroporationmachine and following the manufacturer's guidelines for use. Afterelectroporation, the ES cells are typically allowed to recover undersuitable incubation conditions. The cells are then screened for thepresence of the knockout construct.

Screening can be accomplished using a variety of methods. Where themarker gene is an antibiotic resistance gene, for example, the ES cellsmay be cultured in the presence of an otherwise lethal concentration ofantibiotic. Those ES cells that survive have presumably integrated theknockout construct. If the marker gene is other than an antibioticresistance gene, a Southern blot of the ES cell genomic DNA can beprobed with a sequence of DNA designed to hybridize only to the markersequence Alternatively, PCR can be used. Finally, if the marker gene isa gene that encodes an enzyme whose activity can be detected (e.g.,b-galactosidase), the enzyme substrate can be added to the cells undersuitable conditions, and the enzymatic activity can be analyzed. Oneskilled in the art will be familiar with other useful markers and themeans for detecting their presence in a given cell. All such markers arecontemplated as being included within the scope of the teaching of thisinvention.

The knockout construct may integrate into several locations in the EScell genome, and may integrate into a different location in each EScell's genome due to the occurrence of random insertion events. Thedesired location of insertion is in a complementary position to the DNAsequence to be knocked out, e.g., the Delta3 coding sequence,transcriptional regulatory sequence, etc. Typically, less than about1-5% of the ES cells that take up the knockout construct will actuallyintegrate the knockout construct in the desired location. To identifythose ES cells with proper integration of the knockout construct, totalDNA can be extracted from the ES cells using standard methods. The DNAcan then be probed on a Southern blot with a probe or probes designed tohybridize in a specific pattern to genomic DNA digested with particularrestriction enzyme(s). Alternatively, or additionally, the genomic DNAcan be amplified by PCR with probes specifically designed to amplify DNAfragments of a particular size and sequence (i.e., only those cellscontaining the knockout construct in the proper position will generateDNA fragments of the proper size).

After suitable ES cells containing the knockout construct in the properlocation have been identified, the cells can be inserted into an embryo.Insertion may be accomplished in a variety of ways known to the skilledartisan, however a preferred method is by microinjection. Formicroinjection, about 10-30 cells are collected into a micropipet andinjected into embryos that are at the proper stage of development topermit integration of the foreign ES cell containing the knockoutconstruct into the developing embryo. For instance, as the appendedExamples describe, the transformed ES cells can be microinjected intoblastocytes.

The suitable stage of development for the embryo used for insertion ofES cells is very species dependent, however for mice it is about 3.5days. The embryos are obtained by perfusing the uterus of pregnantfemales. Suitable methods for accomplishing this are known to theskilled artisan, and are set forth by, e.g., et al. (1986) CurrentTopics in Devel. Biol. 20:357-371.

While any embryo of the right stage of development is suitable for use,preferred embryos are male. In mice, the preferred embryos also havegenes coding for a coat color that is different from the coat colorencoded by the ES cell genes. In this way, the offspring can be screenedeasily for the presence of the knockout construct by looking for mosaiccoat color (indicating that the ES cell was incorporated into thedeveloping embryo). Thus, for example, if the ES cell line carries thegenes for white fur, the embryo selected will carry genes for black orbrown fur.

After the ES cell has been introduced into the embryo, the embryo may beimplanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

Offspring that are born to the foster mother may be screened initiallyfor mosaic coat color where the coat color selection strategy (asdescribed above, and in the appended examples) has been employed. Inaddition, or as an alternative, DNA from tail tissue of the offspringmay be screened for the presence of the knockout construct usingSouthern blots and/or PCR as described above. Offspring that appear tobe mosaics may then be crossed to each other, if they are believed tocarry the knockout construct in their germ line, in order to generatehomozygous knockout animals. Homozygotes may be identified by Southernblotting of equivalent amounts of genomic DNA from mice that are theproduct of this cross, as well as mice that are known heterozygotes andwild type mice.

Other means of identifying and characterizing the knockout offspring areavailable. For example, Northern blots can be used to probe the mRNA forthe presence or absence of transcripts encoding either the gene knockedout, the marker gene, or both. In addition, Western blots can be used toassess the level of expression of the Delta3 gene knocked out in varioustissues of the offspring by probing the Western blot with an antibodyagainst the particular Delta3 protein, or an antibody against the markergene product, where this gene is expressed. Finally, in situ analysis(such as fixing the cells and labeling with antibody) and/or FACS(fluorescence activated cell sorting) analysis of various cells from theoffspring can be conducted using suitable antibodies to look for thepresence or absence of the knockout construct gene product.

Yet other methods of making knock-out or disruption transgenic animalsare also generally known. See, for example, Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Recombinase dependent knockouts can also be generated, e.g., byhomologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a Delta-gene can becontrolled by recombinase sequences (described infra).

Animals containing more than one knockout construct and/or more than onetransgene expression construct are prepared in any of several ways. Thepreferred manner of preparation is to generate a series of mammals, eachcontaining one of the desired transgenic phenotypes. Such animals arebred together through a series of crosses, backcrosses and selections,to ultimately generate a single animal containing all desired knockoutconstructs and/or expression constructs, where the animal is otherwisecongenic (genetically identical) to the wild type except for thepresence of the knockout construct(s) and/or transgene(s).

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

Electronic Apparatus Readable Media and Arrays

Electronic apparatus readable media comprising the SPOIL molecules ofthe present invention is also provided. As used herein, “electronicapparatus readable media” refers to any suitable medium for storing,holding or containing data or information that can be read and accesseddirectly by an electronic apparatus. Such media can include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as compactdisc; electronic storage media such as RAM, ROM, EPROM, EEPROM and thelike; general hard disks and hybrids of these categories such asmagnetic/optical storage media. The medium is adapted or configured forhaving recorded thereon a marker of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe SPOIL molecules of the present invention.

A variety of software programs and formats can be used to store themarker information of the present invention on the electronic apparatusreadable medium. For example, the nucleic acid sequence corresponding tothe SPOIL molecules can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicroSoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like, as wellas in other forms. Any number of dataprocessor structuring formats(e.g., text file or database) may be employed in order to obtain orcreate a medium having recorded thereon the SPOIL molecules of thepresent invention.

By providing the SPOIL molecules of the invention in readable form, onecan routinely access the marker sequence information for a variety ofpurposes. For example, one skilled in the art can use the nucleotide oramino acid sequences of the present invention in readable form tocompare a target sequence or target structural motif with the sequenceinformation stored within the data storage means. Search means are usedto identify fragments or regions of the sequences of the invention whichmatch a particular target sequence or target motif.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a bone metabolism disorder, an inflammatory disorder, or an immunedisorder or a pre-disposition to a bone metabolism disorder, aninflammatory disorder, or an immune disorder, wherein the methodcomprises the steps of determining the presence, absence, or aberrantproduction of a SPOIL molecule or a SPOIL variant and based on thepresence, absence or aberrant production of a SPOIL molecule or a SPOILvariant, determining whether the subject has a bone metabolism disorder,an inflammatory disorder, or an immune disorder or a pre-disposition toa bone metabolism disorder, an inflammatory disorder, or an immunedisorder and/or recommending a particular treatment for the bonemetabolism disorder, an inflammatory disorder, or an immune disorder.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a bone metabolism disorder, an inflammatory disorder, or an immunedisorder or a pre-disposition to a bone metabolism disorder, aninflammatory disorder, or an immune disorder, wherein the methodcomprises the steps of determining the presence, absence, or aberrantproduction of a SPOIL molecule or a SPOIL variant and based on thepresence, absence or aberrant production of a SPOIL molecule or a SPOILvariant, determining whether the subject has a bone metabolism disorder,an inflammatory disorder, or an immune disorder or a pre-disposition toa bone metabolism disorder, an inflammatory disorder, or an immunedisorder and/or recommending a particular treatment for the bonemetabolism disorder, an inflammatory disorder, or an immune disorder.The method may further comprise the step of receiving phenotypicinformation associated with the subject and/or acquiring from a networkphenotypic information associated with the subject.

The present invention also provides in a network, a method fordetermining whether a subject has a hematological disorder or apre-disposition to a bone metabolism disorder, an inflammatory disorder,or an immune disorder or a pre-disposition to a bone metabolismdisorder, an inflammatory disorder, or an immune disorder associatedwith the SPOIL molecules, said method comprising the steps of receivinginformation associated with the SPOIL molecule, receiving phenotypicinformation associated with the subject, acquiring information from thenetwork corresponding to the SPOIL molecule and its associateddisorders, and based on one or more of the phenotypic information, theSPOIL molecule, and the acquired information, determining whether thesubject has a bone metabolism disorder, an inflammatory disorder, or animmune disorder or a pre-disposition to a bone metabolism disorder, aninflammatory disorder, or an immune disorder. The method may furthercomprise the step of recommending a particular treatment for the bonemetabolism disorder, the inflammatory disorder, or the immune disorderor a pre-disposition to the bone metabolism disorder, the inflammatorydisorder, or the immune disorder.

The present invention also provides a business method for determiningwhether a subject has a bone metabolism disorder, an inflammatorydisorder, or an immune disorder or a pre-disposition to a bonemetabolism disorder, an inflammatory disorder, or an immune disorder,said method comprising the steps of receiving information associatedwith the SPOIL molecules, receiving phenotypic information associatedwith the subject, acquiring information from the network correspondingto the SPOIL molecules and its associated disorders, and based on one ormore of the phenotypic information, the SPOIL molecule, and the acquiredinformation, determining whether the subject has a bone metabolismdisorder, an inflammatory disorder, or an immune disorder or apre-disposition to a bone metabolism disorder, an inflammatory disorder,or an immune disorder. The method may further comprise the step ofrecommending a particular treatment for the bone metabolism disorder,the inflammatory disorder, or the immune disorder or a pre-dispositionto the bone metabolism disorder, the inflammatory disorder, or theimmune disorder.

The invention also includes an array comprising a SPOIL molecule of thepresent invention. The array can be used to assay expression of one ormore genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression. This allows a profile to bedeveloped showing a battery of genes specifically expressed in one ormore tissues.

In addition to such qualitative determination, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined. Such a determination is useful,for example, to know the effect of cell-cell interaction at the level ofgene expression. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of hematological disorder, progression of hematologicaldisorder, and processes, such a cellular transformation associated withhematological disorder.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing areincorporated herein by reference.

EXAMPLES Example 1 Isolation of a Full-Length cDNA Encoding Human Delta3

Human microvascular endothelial cells (HMVEC catalog #CC2543; Clonetics,San Diego, Calif.) were separated into four samples of cells which weretreated as follows. The first sample was untreated. The second samplewas treated with human TGF-β1 (hTGF-β1) (10 ng/ml) (UpstateBiotechnology, Lake Placid, N.Y., Catalog NO: 01-134). The third samplewas treated with bFGF (10 ng/ml)/VEGF (25 ng/ml) (Upstate Biotechnology,Lake Placid, N.Y., Catalog NO: 01-134, Catalog Nos. 01-106 and 01-185,respectively). The fourth sample was differentiated on Matrigel(Collaborative Biomedical Products, Becton Dickinson Labware, Bedford,Mass.). Cells were treated as indicated for 24 hours, the 4 samples werepooled, and RNA was extracted from the pooled cells using a QIAGENRNeasy kit. The resulting cDNA library was subjected to high throughputrandom sequencing. This allowed identification of a cDNA fragmentcomprising the following 171 nucleotide long sequence:

(SEQ ID NO: 21) GCCCAGGCNGACCCTGGTGTGGACTGTGAGCTGGAGCTCAGCGAGTGTGACAGCAACCCCTGTCGCANTGGAGGCAGCTGTAAGGACCANGAGGATGGCTACCACTGCCTGTGTCCTCCGGGCTACTACGGCNTGCATCGTGAACACNGCACCTCTTAGCTGNGCCGACTC.

Comparison of the nucleotide sequence of this partial cDNA with thesequences in GenBank using the BLAST program (Altschul et al. (1990) J.Mol. Biol. 215:403) revealed that the nucleotide sequence encoded aprotein fragment having a significant homology to Delta proteins. Infact, the amino acid sequence had significant homology with a chickenDelta1 protein (GenBank Accession NO: U26590), a Xenopus Delta1 protein(GenBank Accession NO: L42229), a rat Delta1 protein (GenBank AccessionNO: U78889), a Xenopus Delta2 protein (GenBank Accession NO: U70843) aswell as Notch proteins.

A full-length cDNA of about 3.2 kb was then isolated by screening ahuman microvascular endothelial cell (HMVEC) cDNA library using thepartial cDNA (SEQ ID NO:21). This nucleic acid was deposited at theAmerican Type Culture Collection (ATCC®) on Mar. 5, 1997, and has beenassigned ATCC® Accession NO: 98348. The nucleotide sequence of the cDNAisolated is shown in SEQ ID NO:1.

A nucleic acid sequence comparison of SEQ ID NO:1 against EST sequencedatabases using the BLAST program (Altschul et al. (1990) J. Mol. Biol.215:403) indicated that 5 ESTs have a homology to portions of SEQ IDNO:1. These are all located 3′ of the nucleotide sequence encoding thetransmembrane domain, i.e., downstream of nucleotide 1996 of SEQ IDNO:1. Three of these ESTs (having accession Nos. T33770, T33811, andT07963) have a nucleotide sequence starting at about nucleotide 2044 ofSEQ ID NO:1. However, the nucleotide sequence of the three EST issignificantly different from the nucleotide sequence of hDelta3 in aboutthe first 50 nucleotides 3′ of nucleotide 2044 of SEQ ID NO:1. Two ESTs(having Accession Nos. R32717 and T07962) are located further downstreamof the three ESTs.

The nucleic acid having SEQ ID NO:1 encodes a protein of 685 amino acidshaving SEQ ID NO:2. A comparison of the amino acid sequence of SEQ IDNO:2 with sequences in GenBank using BLASTP (Altschul et al. (1990) J.Mol. Biol. 215:403) reveals that this protein has a certain homology topreviously described Delta proteins. An alignment of the human Delta3protein having SEQ ID NO:2 with the amino acid sequence of mouse Delta1protein (Accession NO: X80903), rat Delta1 protein (Accession NO:U78889), chicken Delta1 protein (Accession NO: U26590), two XenopusDelta1 proteins (Accession Nos. L42229 and U70843) and Drosophila Delta1protein (Accession NO: AA142228) indicates that human Delta3 protein hasthe general structure of a Delta3 protein. In particular, human Delta3protein has a signal peptide corresponding to about amino acid 1 toabout amino acid 17 of SEQ ID NO:2, a DSL motif corresponding to thesequence from about amino acid 173 to about amino acid 217, a firstEGF-like domain corresponding to the sequence from about amino acid 222to about amino acid 250, a second EGF-like domain corresponding to thesequence from about amino acid 253 to about amino acid 281, a thirdEGF-like domain corresponding to the sequence from about amino acid 288to about amino acid 321, a fourth EGF-like domain corresponding to thesequence from about amino acid 328 to about amino acid 359, a fifthEGF-like domain corresponding to the sequence from about amino acid 366to about amino acid 399, a sixth EGF-like domain corresponding to thesequence from about amino acid 411 to about amino acid 437, a seventhEGF-like domain corresponding to the sequence from about amino acid 444to about amino acid 475, an eight EGF-like domain corresponding to thesequence from about amino acid 484 to about amino acid 517, atransmembrane domain corresponding to the sequence from about amino acid530 to about amino acid 553, and a cytoplasmic domain corresponding tothe sequence from about amino acid 554 to about amino acid 685 of SEQ IDNO:2.

An amino acid and nucleotide sequence comparison between the members ofthe Delta1 and Delta3 protein family and human Delta3 on one hand andbetween the members of the Delta1 family reveals that the homologybetween the Delta3 family members is stronger than the homology betweenhuman Delta3 and any of the Delta1 family members. For example, althoughhDelta3 is only approximately 58% similar to the Drosophila Delta1protein; approximately 70% similar to the mouse Delta1 protein;approximately 70% similar to the rat Delta1 protein; approximately 68%similar to the chick Delta1 protein; and approximately 68% similar tothe Xenopus Delta1 proteins; the drosophila, mouse, rat, chick andXenopus Delta1 proteins are very similar to each other (e.g., the mouseand rat Delta1 are about 96% similar to each other). Published PCTapplication WO97/01571 discloses a partial nucleotide and amino acidsequence of a protein having significant homology to Delta1 familymembers, indicating that it is likely to be a human Delta1 protein. Thehomology between the partial amino acid sequence of human Delta1 and theamino acid sequence of human Delta3 is indicated in Table I and showsthat the proteins are encoded by different genes. All these amino acidand nucleotide sequence comparisons indicate that human Delta3 is anadditional species of Delta proteins, sharing some sequence andstructure homology with the Delta1 proteins.

In one embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 455 is a guanine (G) (SEQ ID NO:1). In thisembodiment, the amino acid at position 40 is glutamate (E) (SEQ IDNO:2). In another embodiment of a nucleotide sequence of human Delta3,the nucleotide at position 455 is a cytosine (C) (SEQ ID NO:27). In thisembodiment, the amino acid at position 40 is glutamine (Q) (SEQ IDNO:28). In another embodiment of a nucleotide sequence of human Delta3,the nucleotide at position 455 is a thymidine (T) (SEQ ID NO:29). Inthis embodiment, the amino acid at position 40 is a stop codon (SEQ IDNO:30). In another embodiment of a nucleotide sequence of human Delta3,the nucleotide at position 455 is a adenine (A) (SEQ ID NO:31). In thisembodiment, the amino acid at position 40 is lysine (K) (SEQ ID NO:32).

In one embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 786 is an cytosine (C) (SEQ ID NO:1). In thisembodiment, the amino acid at position 150 is a alanine (A) (SEQ IDNO:2). In an alternative embodiment, a species variant of human Delta3has a nucleotide at position 786 which is a thymidine (T) (SEQ IDNO:33). In this embodiment, the amino acid at position 150 is valine (V)(SEQ ID NO:34), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of human Delta3, thenucleotide at position 594 is a cytosine (C) (SEQ ID NO:1). In thisembodiment, the amino acid at position 86 is threonine (T) (SEQ IDNO:2). In an alternative embodiment, a species variant of human Delta3has a nucleotide at position 594 which is a guanine (G) (SEQ ID NO:35).In this embodiment, the amino acid at position 86 is serine (S) (SEQ IDNO:36), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of human Delta3, wherein thenucleotide at position 883 is a thymidine (T) (SEQ ID NO:1). In thisembodiment, the amino acid at position 182 is aspartate (D) (SEQ IDNO:2). In an alternative embodiment, a species variant of human Delta3has a nucleotide at position 883 which is an adenine (A) (SEQ ID NO:37).In this embodiment, the amino acid at position 182 is glutamate (E) (SEQID NO:38), i.e., a conservative substitution.

Example 2 Isolation of a Full-Length cDNA Encoding Mouse Delta3

A mouse Delta3 cDNA was identified from mouse lung database library ofexpressed sequences using the human Delta3 cDNA (SEQ ID NO:1) as a querysequence. The most homologous sequence, SEQ ID NO:24 was identified as a3.2 kb cDNA.

The nucleic acid having SEQ ID NO:24 encodes a protein of 686 aminoacids having the amino acid sequence shown in SEQ ID NO:25. An alignmentof the human and mouse Delta3 proteins having SEQ ID NO:2 and 25,respectively, indicates that human and mouse Delta3 proteins havesignificant similarity and identity (i.e., 88.2% similar and 86.6%identical) suggesting evolutionary conservation due to an essentialbiological function.

Mouse Delta3 protein has a signal peptide corresponding to about aminoacid 1 to about amino acid 17 of SEQ ID NO:25, a DSL motif correspondingto the sequence from about amino acid 174 to about amino acid 218, afirst EGF-like domain corresponding to the sequence from about aminoacid 223 to about amino acid 251, a second EGF-like domain correspondingto the sequence from about amino acid 254 to about amino acid 282, athird EGF-like domain corresponding to the sequence from about aminoacid 289 to about amino acid 322, a fourth EGF-like domain correspondingto the sequence from about amino acid 329 to about amino acid 360, afifth EGF-like domain corresponding to the sequence from about aminoacid 367 to about amino acid 400, a sixth EGF-like domain correspondingto the sequence from about amino acid 412 to about amino acid 438, aseventh EGF-like domain corresponding to the sequence from about aminoacid 445 to about amino acid 476, an eight EGF-like domain correspondingto the sequence from about amino acid 485 to about amino acid 518, atransmembrane domain corresponding to the sequence from about amino acid531 to about amino acid 554, and a cytoplasmic domain corresponding tothe sequence from about amino acid 555 to about amino acid 686 of SEQ IDNO:25.

In one embodiment of a nucleotide sequence of mouse Delta3, thenucleotide at position 49 is cytosine (C) (SEQ ID NO:24). In thisembodiment, the amino acid at position 4 is alanine (A) (SEQ ID NO:25).In an alternative embodiment, a species variant of mouse Delta3 has anucleotide at position 49 which is thymidine (T) (SEQ ID NO:39). In thisembodiment, the amino acid at position 4 is valine (V) (SEQ ID NO:40),i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, thenucleotide at position 51 is thymidine (T) (SEQ ID NO:24). In thisembodiment, the amino acid at position 5 is serine (S) (SEQ ID NO:25).In an alternative embodiment, a species variant of mouse Delta3 has anucleotide at position 51 which is a adenine (A) (SEQ ID NO:41). In thisembodiment, the amino acid at position 5 is threonine (T) (SEQ IDNO:42), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, thenucleotide at position 109 is guanine (G) (SEQ ID NO:24). In thisembodiment, the amino acid at position 24 is arginine (R) (SEQ IDNO:25). In an alternative embodiment, a species variant of mouse Delta3has a nucleotide at position 109 which is adenine (A) (SEQ ID NO:43). Inthis embodiment, the amino acid at position 24 is histidine (H) (SEQ IDNO:44), i.e., a conservative substitution.

In one embodiment of a nucleotide sequence of mouse Delta3, wherein thenucleotide at position 130 is a thymidine (T) (SEQ ID NO:24). In thisembodiment, the amino acid at position 31 is phenylalanine (F) (SEQ IDNO:25). In an alternative embodiment, a species variant of mouse Delta3has a nucleotide at position 130 which is adenine (A) (SEQ ID NO:45). Inthis embodiment, the amino acid at position 31 is tyrosine (Y) (SEQ IDNO:46), i.e., a conservative substitution.

Example 3 Tissue Expression of the hDelta3 Gene

This Example describes the tissue distribution of Delta3 protein, asdetermined by Northern blot hybridization with a 1.6 kb fragment ofhuman Delta3 cDNA corresponding to the extreme 3′ end of SEQ ID NO:1 andby in situ hybridization using a probe complementary to nucleotides1290-1998 of SEQ ID NO:1.

Northern blot hybridizations with the various RNA samples were performedunder standard conditions and washed under stringent conditions, i.e.,in 0.2×SSC at 65° C. In each sample, the probe hybridized to a singleRNA of about 3.5 kb. The results of hybridization of the probe tovarious mRNA samples are described below.

Hybridization of a Clontech Fetal Multiple Tissue Northern (MTN) blot(Clontech, LaJolla, Calif.) containing RNA from fetal brain, lung,liver, and kidney indicated the presence of Delta3 RNA in each of thesefetal tissues. Expression was significantly higher in fetal lung andkidney than in fetal brain and liver. Hybridization of a Clontech humanMultiple Tissue Northern I (MTNI) and Multiple Tissue Northern H (MTNII)blots (Clontech, LaJolla, Calif.) containing RNA from adult heart,brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, mucosal lining of thecolon, and peripheral blood leukocytes with the human 1.6 kb Delta3probe indicated expression in heart, placenta, lung, skeletal muscle,kidney, pancreas, spleen, thymus, prostate, testis, ovary, smallintestine and colon. Expression was particularly strong in adult heart,placenta, lung, and skeletal muscle. Expression was also found in adultbrain, liver and testis. However, no significant amount of hDelta3 mRNAwas detected in adult peripheral blood leukocytes.

Further, Northern blot hybridization of total mRNA from HMVEC cellstreated with TGF-β1 at 10 ng/ml for 24 hours, bFGF at 10 ng/ml/VEGF at25 ng/ml for 24 hours, or untreated for 24 hours indicated that Delta3expression was induced upon induction with bFGF/VEGF. Accordingly,expression of Delta3 is up-regulated in HMV endothelial cells inresponse to certain growth factors.

Hybridization of a “cancer” Northern blot containing RNA from HL-60,HeLa, K562, MoLT4, Raji, SW480, A549, and G361 cells, revealed thatDelta3 is expressed at high levels in the colorectal carcinoma cell lineSW480. Thus, Delta3 expression is high in at least certain tumor cells.

Delta-3 in situs on paraffin embedded mouse embryos were performed.Expression was seen in endothelial cells of the secondary vasculatureand in preendothelial cells in the bone marrow. There is no expressionin endothelial cells after day P 1.5.

For in situ hybridization analysis of mDelta3, 10 m sagittal sections offresh frozen day E13.5, E14.5, E15.5, E16.5, E18.5 and P1.5 embryos ofB6 mice, as well as 8 m cross sections of brain, spinal cord, eye andharderian gland, submandibular gland, white fat, stomach, heart, lung,liver, spleen, thymus, small intestine, lymph node, pancreas, skeletalmuscle, testes, ovary, placenta, kidney and adrenal gland from adult B6mice were used for hybridization. Sections were postfixed with 4%formaldehyde in DEPC-treated 1× phosphate-buffered saline at roomtemperature for 10 minutes before being rinsed twice in DEPC-treated 1×phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH8.0).Following incubation in 0.25% acetic anhydride-0.1 M triethanolamine-HClfor 10 minutes, sections were rinsed in DEPC-treated 2×SSC (1×SSC is0.15M NaCl plus 0.015M sodium citrate). Tissue was dehydrated through aseries of ethanol washes, incubated in 100% chloroform for 5 minutes,and then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1minute and allowed to air dry.

The hybridization was performed using a ³⁵S-radiolabeled cRNA probe fromthe DNA sequence of nucleotides 1290-1998 of SEQ ID NO:1.

Tissues were incubated with probe (approximately 5×10⁷ cpm/ml) in thepresence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mMEDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeasttotal RNA type X1, 1×Denhardt's solution, 50% formamide, 10% dextransulfate, 100 mM dithiothreitol, 0.1% sodium dodecylsulfate (SDS), and0.1% sodium thiosulfate for 18 h at 55 C.

After hybridization, slides were washed with 2×SSC. Sections were thensequentially incubated at 37 C in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 ug of RNase A per ml for 30 minutes, and finally in TNE forminutes. Slides were then rinsed with 2×SSC at room temp, washed with2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1 hour,and 0.2×SSC at 60° C. for 1 hour. Sections were then dehydrated rapidlythrough serial ethanol-0.3 M sodium acetate concentrations before beingair dried and exposed to Kodak Biomax MR scientific imaging film for 6days at room temperature.

Expression was most abundant and wide spread during embryogenesis.Strongest expression was observed in the eye in all of the embryonicages tested. Signal in a pattern suggestive of neuronal expression wasnot observed in any other tissues making the expression in the eyeunique. Moderate ubiquitous expression was also detected in lung, thymusand brown fat during embryogenesis. A multifocal, scattered signal wasalso observed throughout the embryo. This signal pattern was morefocused in the cortical region of the kidney and outlining theintestinal tract. Adult expression was highest in the ovary and thecortical regions of the kidney and adrenal gland.

Thus, Delta3 is expressed in numerous tissues, but is not detected incertain tissues, e.g., peripheral blood leukocytes and adult hearttissue (at least when using Northern blot hybridization), is expressedat relatively high levels in at least some tumor cells, e.g., coloncarcinoma cells, and its expression can be up-regulated in response tosome growth factors, e.g., bFGF and VEGF. Furthermore, in situhybridization shows that mDelta3 is expressed most strongly indeveloping tissues of eye, thymus, lung and brown fat.

A Southern blot containing DNA from a panel of a human/hamstermono-chromosomal somatic cell hybrids was probed with an hDelta1 cDNAprobe. The results obtained clearly indicates that the human Delta3 generesides on chromosome 15.

Example 4 Increased Expression of hDelta3 in Differentiating EndothelialCells

This Example shows that the expression of the hDelta3 gene increases indifferentiating endothelial cells relative to non-differentiatingendothelial cells.

HMVEC cells were separated into 5 cultures and treated as follows: (1)cells were induced to quiescence by growth in basal endothelial growthmedium (EGM) (Clontech) which contains 10% fetal calf serum (FCS); (2)cells were grown in complete endothelial growth medium (EGM-MV)(Clontech, Catalog NO: CC-3125) which contains 10% FCS and growthfactors; (3) cells were stimulated to proliferate by culture in EGM-MVin the presence of bFGF at 10 ng/ml and VEGF at 25 ng/ml; (4) cells werestimulated to proliferate by culture in EGM-MV in the presence ofTGF-131 at 10 ng/ml; and (5) cells were stimulated to differentiate byculture in EGM-MV on Matrigel. After 24 hours of culture, the cells wereharvested, the RNA was extracted and submitted to Northern blotanalysis. Hybridization was performed with the 1.6 kb hDelta3 probedescribed above. The results indicate that among the culture conditionstested, quiescent cells express the lowest amount of hDelta3 (at abarely detectable level). Cells which are proliferating express a higherlevel of hDelta3. Interestingly, the mRNA level of hDelta3 was stronglyincreased in cells induced to differentiate by plating on Matrigel.

Thus, this Example clearly demonstrates that hDelta3 expression isstrongly increased in cells induced to differentiate and also in cellsinduced to proliferate.

Example 5 hDelta3 is Located in a Chromosomal Region Associated withACCPN

The location of hDelta3 on human chromosome 15 was determined usingradiation hybrid (RH) mapping.

A sequence tagged site (STS) was generated from the 3′ untranslatedregion of the gene using a forward primer having the nucleotide sequenceGTTTACATTGCATCCTGGAT (SEQ ID NO:51) and a reverse primer having thenucleotide sequence CTCTTCTGTTCCTCTGGTTG (SEQ ID NO:22). The STS wasused to screen the Genebridge 4 (Gyapay et al. (1996) Human MolecularGenetics 5:339) and the Standford G3 (Stewart et al. (1997) Genome Res.7:422) radiation hybrid panels. These panels were derived by fusion ofirradiated human donor cells with rodent recipient cells and can be usedfor positioning STS markers within existing framework maps, orderingmarkers in the region of interest as well as establishing the distancebetween markers.

RH mapping was performed by PCR under the following conditions: 25 ngDNA/20 μl reaction, 0.5 of each primer, 0.2 mM of each nucleotide, 1.5mM MgCl₂, 1× buffer as provided by the manufacturer of the enzyme, 35cycles at 94° C., 55° C., 72° C. for 30 seconds each.

The results of the RH mapping indicated that hDelta3 maps to 15q12-15close to framework marker D15S1244 on the Stanford G3 panel and close toframework marker D15S144 on the Genebridge 4 panel with a LOD score >3.Searching of the OMIM database (Online Mendelian Inheritance in Man)indicated that this region has previously been genetically linked to aneurological disorder called Agenesis of the Corpus Callosum withPeripheral Neuropathy (ACCPN) (Casaubon et al. (1996) Am. J. Hum. Genet.58:28).

Example 6 Delta3 Encodes a Notch Ligand

The example presented herein demonstrates that Delta3 encodes a NotchLigand. In particular, the data presented herein shows, first, thathDelta3 encodes a functional Notch ligand as determined by its abilityto block differentiation of C2C12 cells. When C2C12 cells areco-cultured, under low mitogenic conditions, with NIH3T3 cellsexpressing a Notch ligand, the differentiation to myotubes by the C2C12cells is blocked. (Lindsell et al. (1995) Cell 80:909). If the cellsdifferentiate troponin T is expressed, if differentiation is blocked notroponin T expression is seen. In addition, the data presented hereindirectly demonstrates that Delta3 binds Notch one and Notch 2. Third,the data presented in this section identifies several cell types thatendogenously exhibit Delta3 receptors.

To determine whether hDelta3 in fact encodes a functional Notch ligand,NIH3T3 cells were engineered to express hDelta3, co-cultured with C2C12cells and analyzed for troponin T expression. Briefly, NIH3T3 cells wereinfected with a retrovirus containing the hDelta3 coding region clonedinto the MIGR retroviral vector (Pear et al. (1998) Blood 92:3780). Thisvector contains an Internal Ribosome Entry Site (IRES) downstream of thecloning site, followed by the cDNA for the green fluorescent protein(GFP). GFP expression from the vector is monitored to assess theefficiency of transduction of the vector into the target cells. C2C12cells were plated in 10 cm dishes and cultured in DMEM media with 10%

Inactivated Fetal Calf Serum (10% IFS) until 70% confluent. C2C12 cellswere then washed 1× with PBS. 5×10⁶ NIH3T3 cells harboring either anempty vector, a vector expressing hDelta3 or a vector expressingJagged-1 were resuspended in 10 mls DMEM media containing 10% Horseserum (10% HS), and laid on top of the C2C12 cells.

Control experiments involved the solitary culture of C2C12 cells indifferentiation media (10% HS) as well as in growth media (10% IFS). Thewhole population of cells was lysed three to four days later and equalamounts of protein was resolved on an SDS-polyacrylamide gel. Theproteins were then transferred onto a nitrocellulose membrane and probedwith an anti-Troponin T antibody (Sigma, 1:200). A secondary incubationwith an anti-mouse antibody conjugated to horseradish peroxidase allowedfor detection using chemiluminescence reagents (Amersham). When cellsNIH3T3 cells containing the empty vector were co-cultured with C2C12cells, troponin T was expressed, indicating that the C2C12 cells hadindeed differentiated into myotubes. When NIH3T3 cells expressinghDelta3 were co-cultured with C2C12 cells, no expression of troponin Twas seen, indicating that C2C12 differentiation was blocked by hDelta3.This result is similar to that seen when NIH3T3 cells expressing Jagged1 (a functional Notch ligand).

Next, Delta3 was tested for its ability to bind human notch1 and notch2.293T cells were transiently transfected with expression plasmids (PCMVpoly-neo) encoding full-length Notch1 or Notch2. Two days aftertransfection cells were incubated with purified protein consisting ofthe extracellular domain of hDelta3 fused in frame to the Fc portion ofimmunoglobulin G (hDelta3-Fc) at 10 μg/ml or with control proteinconsisting of human immunoglobulin G1 (hIgG1) at 10 ug/ml in stainingbuffer (PBS containing 3% fetal calf serum, 1 mM CaCl₂ and 0.02% sodiumazide). After one hour of incubation, cells were washed three times instaining buffer and bound protein was detected by incubating the cellswith FITC-conjugated anti-human IgG for 30 minutes. Cells were analyzedunder fluorescence microscopy.

Binding of hDelta3-Fc fusions to cells expressing Notch1 and Notch2 butnot to cells transfected with empty expression vector, was detected.Binding was calcium-dependent and was abolished in the presence of 5 mMEDTA. Control-Fc fusions and hIgG1 did not show any binding totransfected cells. These results establish hDelta3 as a ligand forNotch1 and Notch2 and show that the extracellular domain of hDelta3 issufficient to mediate binding to Notch.

Therefore, Delta3, including hDelta3, represents a polypeptide which canfunction as a bona fide Notch ligand.

Next, Cell lines were tested for the presence of an endogenous receptorfor hDelta3. Briefly, cells were washed two times in staining buffer andincubated with hDelta3-Fc or hIgG1 (10 μg/ml in staining buffer) at acell concentration of 5×10⁶ per ml. After an incubation of one hour onice, cells were washed three times in staining buffer and bound proteinwas detected by incubating the cells with FITC-conjugated secondaryantibody (anti-human hIgG1) for 30 min on ice. Cells were analyzed byflow cytometry on a FACSCalibur. Binding of hDelta3-Fc was seen toJurkat, 32D, C2C12 and Cos cells. A control Fc fusion protein and hIgG1did not show binding to these cell lines. The binding of hDelta3-Fc wasdependent on calcium since the binding was abolished by the addition of5 mM EDTA to the binding buffer.

Example 7 Delta3 Affects Early Development and Muscle CellDifferentiation

The data presented herein demonstrate that among the roles of Delta3 isa function that involves early development and muscle celldifferentiation.

Materials and Methods

Preparation of hDelta3 RNA: The template for the hDelta3 in vitrotranscription reaction was prepared from the DNA construct containingthe hDelta3 sequence inserted in a pCS2++ vector, which was thenlinearized using AscI. Capped RNA was synthesized using SP6 RNApolymerase from the linearized plasmid using mMESSAGE mMACHINE kit(Ambion, Austin, Tex.) according to the manufacturer's instructions. Invitro transcribed capped RNA was purified using RNAesy kit (Qiagen) andanalyzed by gel electrophoresis.

HDelta3 RNA injection into Xenopus embryos: Xenopus embryos wereobtained by in vitro fertilization, dejellied in 2% cysteine HCl (pH7.6), washed thoroughly in Modified Ringer's solution, and incubated at15-25° C. Embryos were transferred to injection solution (ModifiedRinger's solution containing 3% Ficoll) prior to injections. One ng and2.5 ng of hDelta3 RNA were injected into one blastomere at the 2-cellstage. Embryos were transferred to 0.1×MMR from the injection solutionafter approximately 6 hours and grown until the appropriate stage.

Embryos for histological examination were fixed in 4% formaldehyde inPBS overnight, embedded in paraffin and stained with Heidenhain's Azanstain by standard procedures. Transverse sections of injected embryosshow disruption of somitic organization and somite boundaries on theinjected side.

Xenopus animal cap assay: 2 ng of hDelta3 RNA was injected into theanimal pole of each of the 2 Xenopus blastomeres at the 2-cell stage.Animal caps from uninjected or injected embryos were explanted at stage9 and cultured in 1XModified Ringers containing 0.01% BSA and 50 ug/mlgentamycin. Animal caps were cultured until control embryos have reachedstage 23-24. Animal cap tissue was lysed and total RNA was extractedusing RNeasy kit (Qiagen). RT-PCRs were performed on these samples usinggene-specific primers and appropriate annealing temperatures and theproducts analyzed by gel electrophoresis. The primers used in thisexperiment were specific to genes EF1-alpha, XCG-1, NCAM, Xbra, M-actin,Sox-17 (Amaravadi et al. (1997). Dev. Biol. 192:392-404). RT-PCRanalysis did not indicate expression of any of the specific marker genestested.

Results:

Examination of embryos injected with hDelta3 RNA two days post-injectionshowed an overexpression phenotype involving axial disruption indicativeof an effect on somites and anterior dorsal structures such as eyes andcement glands were not well differentiated in half of the injectedembryos. These results suggest that hDelta3 has an effect on earlytissue development/differentiation.

The differential stain used in this study also indicated an enlargementof somite size on the injected side demonstrating that hDelta3 has aneffect on muscle cells and overexpression can lead to enlarged musclemass. Notch/Delta signaling has been shown to play a key role insomitogenesis/myogenesis in various species (Wittenberger et al., (1999)EMBO J. 18:915-922); Dornseifer et al. (1997) Mech. Dev. 63:159-171);Kusumi et al. (1998) Nat. Genet. 19:274-278).

Therefore, the results presented herein indicate that Delta3 can beinvolved in early development (e.g., can have a role downstream of germlayer specification function), and can be involved in modulatingmyogenesis and muscle cell differentiation.

Example 8 Identification of Delta Therapeutics

This Example describes a simple assay for isolating Delta therapeutics,(e.g., agonist or antagonist of a Delta activity), e.g., Delta3therapeutics. Based at least in part on the results described in theprevious Examples, Delta therapeutics can be used for treating variousdiseases, including neurological diseases, and/or hyper- orhypoproliferative diseases, hematologic disorders, immunodeficiencystates and diseases or conditions associated with defects in vasculatureand/or conditions requiring neovascularization and/or conditionshallmarked by aberrant neovascularization, e.g., diabetic retinopathy.In addition, based at least in part on the similarity of amino acidsequence and structure between the various Delta proteins, Delta3therapeutics can be used to treat diseases or conditions associated withan aberrant Delta3 activity or an aberrant Delta activity other than aDelta3 activity. Similarly, Delta3 therapeutics as well as Deltatherapeutics other than Delta3 therapeutics can be used to treatdiseases or conditions associated with an aberrant Delta3 activity. Theassay set forth below is applicable to Delta proteins other than Delta3proteins.

A Delta3 therapeutic can be identified by using an in vitro assay, inwhich the interaction between a Delta3 protein and a Delta3 bindingprotein, e.g., a Notch protein, is determined in the presence and in theabsence of a test compound. A soluble binding fragment of a Delta3protein can be prepared by expression of the extracellular portion ofhuman Delta3, e.g., about amino acids 1-529 of SEQ ID NO:2 or aboutamino acids 1-530 of SEQ ID NO:25, in E. coli according to methods knownin the art. Alternatively, the Delta3 protein fragment can be aboutamino acid 173 to about amino acid 517 of SEQ ID NO:2 or from aboutamino acid 174 to about amino acid 508 of SEQ ID NO:25. Similarly, aDelta3 binding fragment of a Delta3 binding protein (i.e., Delta3binding partner) can be produced recombinantly.

A Delta3 binding protein can be a Notch protein and can be identified,e.g., by determining whether the protein is capable of binding to aDelta3 protein. A nucleic acid encoding a Notch protein can be obtained,e.g., by PCR amplifying a portion of a Notch gene encoding at least anEGF-like domain, using primers having a nucleotide sequence derived fromthe nucleotide sequence of a Notch gene present in GenBank or disclosedin PCT Application NO: PCT/US92/03651 or PCT/US93/09338.

Test compounds can then be tested to determine whether they inhibit theinteraction between the Delta3 and the Delta3 binding protein by usingan ELISA type assay. Accordingly, one of the recombinantly producedDelta3 protein and the Delta3 binding protein, e.g., Notch protein, isattached to a solid phase surface and the other protein is labeled,e.g., such as by tagging the protein with an epitope, for which anantibody is available (e.g., FLAG epitope, available from InternationalBiotechnologies, Inc.). As a non-limiting example of an assay, theDelta3 protein can be linked to the wells of a microtiter (96 well)plate by overnight incubation of the protein at a concentration of 10μg/ml in PBS. After blocking unoccupied sites on the plate with a BSAsolution, various amounts of test compounds and the recombinantlyproduced Delta3 binding protein are added to the wells in a buffersuitable for a specific interaction between the proteins.

After an incubation time of several hours, the wells are rinsed withbuffer, and the amount of Delta3 binding protein attached to the wellsis determined. The amount of bound protein can be determined byincubating the wells with an anti-tag, e.g., anti-myc, antibody, whichcan then be detected by enzyme immunoassay. The amount of bound proteinis then determined by determining the optical density using an ELISAreader. A lower amount of Delta3 binding protein in a well thatcontained a test compound relative to a well that did not contain a testcompound is indicative that the test compound inhibits the interactionbetween Delta3 and a Delta3 binding protein.

In a further non-limiting example of a binding assay, a recombinantlyproduced and labeled Delta3 polypeptide, or fragment thereof capable ofbinding a Delta3 binding protein, is incubated, with or without a testcompound, with cells expressing the Delta3 binding protein (Shimizu etal. (1999) J. Biol. Chem. 274:32961-32969). Alternatively, therecombinant Delta3 polypeptide is not labeled and is detected uponbinding the cell by a second Delta3 binding protein, such as anantibody. A lower amount of Delta3 binding protein in a well thatcontained a test compound relative to a well that did not contain a testcompound is indicative that the test compound inhibits the interactionbetween Delta3 and a Delta3 binding protein.

A Delta3 therapeutic can also be identified by using a reporter assay inwhich the level of expression of a reporter construct under the controlof a Delta3 promoter is measured in the presence or absence of a testcompound. A Delta3 promoter can be isolated by screening a genomiclibrary with a Delta3 cDNA which preferably contains the 5′ end of thecDNA. A portion of the Delta3 promoter, typically from about 50 to about500 base pairs long is then cloned upstream of a reporter gene, e.g., aluciferase gene, in a plasmid. This reporter construct is thentransfected into cells, e.g., neural cells or endothelial cells.Transfected cells are then be distributed into wells of a multiwellplate and various concentrations of test compounds are added to thewells. After several hours incubation, the level of expression of thereporter construct is determined according to methods known in the art.A difference in the level of expression of the reporter construct intransfected cells incubated with the test compound relative totransfected cells incubated without the test compound will indicate thatthe test compound is capable of modulating the expression of the Delta3gene and is thus a Delta3 therapeutic.

Example 9 Isolation and Characterization of Human FTHMA-070 cDNAs

The nucleic acid molecule encoding FTHMA-070 was identified during thesequencing of clones present in a cardiac coronary artery smooth musclecell library. A clone was identified which appeared to have somehomology to TNF receptor. This clone proved to encode FTHMA-070. Thenucleic acid sequence and deduced amino acid sequence of FTHMA-070,which has homology to tumor necrosis factor receptor (including thedeath domain) are shown in SEQ ID NO:53 and SEQ ID NO:54, respectively.

Example 10 Characterization of FTHMA-070 Proteins

The human FTHMA-070 cDNA isolated as described above (SEQ ID NO:53)encodes a 401 amino acid protein (SEQ ID NO:54). FTHMA-070 is predictedto include a 21 amino acid signal peptide (amino acid 1 to about aminoacid 21 of SEQ ID NO:54) preceding the 380 mature protein (about aminoacid 22 to amino acid 401; SEQ ID NO:56).

Example 11 Preparation of FTHMA-070 Proteins

Recombinant FTHMA-070 can be produced in a variety of expressionsystems. For example, the mature FTHMA-070 peptide can be expressed as arecombinant glutathione-S-transferase (GST) fusion protein in E. coliand the fusion protein can be isolated and characterized. Specifically,as described above, FTHMA-070 can be fused to GST and this fusionprotein can be expressed in E. coli strain PEB199. Expression of theGST-FTHMA-070 fusion protein in PEB 199 can be induced with IPTG. Therecombinant fusion protein can be purified from crude bacterial lysatesof the induced PEB 199 strain by affinity chromatography on glutathionebeads.

Example 12 Isolation and Characterization of Human FTHMA-070 cDNAs

The nucleic acid molecule encoding T85 (originally called FMHB-6D4 andFMHB-SD4) was identified using a screen designed to identify genesencoding proteins having a functional signal sequence. Briefly, alibrary was prepared in which each of the clones contained a human fetalbrain cDNA ligated to a sequence encoding a detectable protein whichlacked a signal sequence. If the human fetal cDNA encodes a functionalsignal sequence, it will permit the secretion and detection of thedetecable protein. This clone library was used to transfect mammaliancells. Clones which secreted the detectable protein were then identifiedand the corresponding human fetal brain cDNA was isolated and sequencedusing standard techniques. In this way it was possible to identify aclone encoding T85. The nucleic acid sequence and deduced amino acidsequence of T85 are shown in SEQ ID NO:57 and SEQ ID NO:58,respectively.

Example 13 Characterization of T85 Proteins

The human T85 cDNA isolated as described above (SEQ ID NO:57) encodes a753 amino acid protein (SEQ ID NO:58). The signal peptide predictionprogram SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6)predicted that T85 includes a 20 amino acid signal peptide (amino acid 1to about amino acid 20 of SEQ ID NO:58) preceding the 733 amino acidmature protein (about amino acid 21 to amino acid 753 of SEQ ID NO:58;SEQ ID NO:60). For general information regarding PFAM identifiers andHidden Markov Model (HMM) consensus sequences refer to Sonnhammer et al.(1997) Protein 28:405-420.

T85 has a two regions (amino acids 525-610 and 638-727 of SEQ ID NO:58;SEQ ID NO:61 and SEQ ID NO:62, respectively) of homology to afibronectin type III domain (based on HMM PF00041; SEQ ID NO:70). Also,T85 has a five regions (amino acids 43-101; 145-203; 237-298; 329-394;and 433-491 of SEQ ID NO:58; SEQ ID NOs:63-67) of homology to a Igsuperfamily domain. T85 also includes an RGD motif starting at aminoacid 247 of SEQ ID NO:58; a cytokine receptor homolgy N-terminal (BC)domain (CC-CC) at amino acids 516-600 of SEQ ID NO:58.

Example 14 Preparation of T85 Proteins

Recombinant T85 can be produced in a variety of expression systems. Forexample, the mature T85 peptide can be expressed as a recombinantglutathione-S-transferase (GST) fusion protein in E. coli and the fusionprotein can be isolated and characterized. Specifically, as describedabove, FTHMA-070 can be fused to GST and this fusion protein can beexpressed in E. coli strain PEB 199. Expression of the GST-T85 fusionprotein in PEB 199 can be induced with IPTG. The recombinant fusionprotein can be purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads.

Example 15

As shown in FIGS. 2A-D, T85 exhibits considerable homology to human Roboprotein (Kidd et al. (1997) Cell 92:205-215), an axon guidance receptorthat is thought to play and important role in neuronal development,specifically, control of midline crossing. Accordingly, T85 may alsoplay a role in neuronal development. Thus, T85 nucleic acids,polypeptides, T85 agonists, and T85 antagonists may be useful in thetreatment of neurological disorders.

Example 16 Isolation and Characterization of Human Tango-77 cDNAs

Cytokine genes IL-1α, IL-1β and IL-1ra have been found to be closelyclustered on chromosome 2, i.e., IL-1α, IL-1β and IL-1ra are locatedwithin 450 kb of each other. BAC clones containing IL-1α and IL-1β wereused to identify other proximal unknown cytokine genes. To do this, aBAC clone containing IL-1α and IL-1β was selected from a BAC library(Research Genetics, Huntsville, Ala.) using specific primers designedagainst IL-1α and IL-1β. The DNA from the BAC was extracted and used tomake a random-sheared genomic library. From this BAC library, 4000clones were selected for sequencing. The resulting genomic sequenceswere then assembled into contigs and used to screen proprietary andpublic data bases. One genomic contig was found to contain two segmentsof sequences which resemble IL-1ra. These two segments are potentialexons of Tango-77 gene.

Two PCR primers were then designed from the two potential exons and usedto screen a panel of cDNA libraries for the expression of a Tango-77message. A cDNA library from TNF-α treated human lung epithelia showed apositive band of the predicted size (i.e., if the two exons are splicedtogether). Using the PCR fragment as a probe, a single cDNA clone wasisolated from the same library. This cDNA contains an insert of 989 bp.The cDNA clone contains three possible open reading frames. The firstopen reading frame encompasses 534 nucleotides (nucleotides 356-889 ofSEQ ID NO:71; SEQ ID NO:73) and encodes a 178 amino acid protein (SEQ IDNO:72). This protein may include a predicted signal sequence of about 63amino acids (from amino acid 1 to about amino acid 63 of SEQ ID NO:72(SEQ ID NO:74)) and a predicted mature protein of about 115 amino acids(from about amino acid 64 to amino acid 178 of SEQ ID NO:72 (SEQ IDNO:75)).

The second putative nucleotide open reading frame encompasses 498nucleotides (nucleotides 389-889 of SEQ ID NO:71; SEQ ID NO:76) andencodes a 167 amino acid protein (SEQ ID NO:77). This protein includes apredicted signal sequence of about 52 amino acids (from amino acid 1 toabout amino acid 52 of SEQ ID NO:77 (SEQ ID NO:78)) and a predictedmature protein of about 115 amino acids (from about amino acid 53 toamino acid 167 of SEQ ID NO:77 (SEQ ID NO:79)).

The third open reading frame (nucleotides 372-889 of SEQ ID NO:71; SEQID NO:80) encompasses 408 nucleotides and encodes a 136 amino acidprotein (SEQ ID NO:81). This protein includes a predicted signalsequence of about 21 amino acids (from amino acid 1 to about amino acid21 of SEQ ID NO:81 (SEQ ID NO:82)) and a predicted mature protein ofabout 115 amino acids (from about amino acid 22 to amino acid 136 of SEQID NO:81 (SEQ ID NO:83)).

Tango-77 is predicted to be 35% identical to human IL-1ra at the aminoacid level.

Example 17 Expression of Tango-77 mRNA in Human Tissues

The expression of Tango-77 was analyzed using Northern blothybridization. A PCR generated 989 bp Tango-77 product was radioactivelylabeled with ³²P-dCTP using the Prime-It kit (Stratagene; La Jolla,Calif.) according to the instructions of the supplier. Filterscontaining human mRNA (MTNI and MTNII: Clontech; Palo Alto, Calif.) wereprobed in ExpressHyb hybridization solution (Clontech) and washed athigh stringency according to manufacturer's recommendations.

Tango-77 mRNA was not detected in any unstimulated tissues (brain,liver, spleen, skeletal muscle, testis, pancreas, heart, kidney andperipheral blood leukocytes) mRNA on Clontech Northern blots.

Over 96 cDNA libraries were then tested for the presence of Tango-77using PCR amplification. Only three libraries displayed a positivesignal. These libraries were the TNFα-treated bronchoepithelium,TNFα-treated SSC cell line and anti-CD3-treated T cells.

Example 18 Characterization of Tango-77 Proteins

In this example, the predicted amino acid sequence of human Tango-77protein was compared to the amino acid sequence of known protein IL-1ra.In addition, the molecular weight of the human Tango-77 proteins waspredicted.

The human Tango-77 cDNA (SEQ ID NO:71) isolated as described aboveencodes a 178 amino acid protein (SEQ ID NO:72) or a 167 amino acidprotein (SEQ ID NO:77) or a 136 amino acid protein (SEQ ID NO:81). Thesignal peptide prediction program SIGNALP Optimized Tool (Nielsen et al.(1997) Protein Engineering 10:1-6) predicted that Tango-77 includes a 63amino acid signal peptide (amino acid 1 to about amino acid 63 of SEQ IDNO:72 (SEQ ID NO:74)) preceding the 115 mature protein; or preceding the115 mature protein (about amino acid 52 to amino acid 167 of SEQ IDNO:77 (SEQ ID NO:78)); or preceding the 115 mature protein (about aminoacid 21 to amino acid 136 of SEQ ID NO:81; SEQ ID NO:82).

As shown in FIG. 3, Tango-77 has a region of homology to IL-1ra (SEQ IDNO:84).

Mature Tango-77 has a predicted MW of about 13 kDa and the predicted MWfor the immature Tango-77 is 19.6 kDa, 18.5 kDa or 15.2 kDa, notincluding post-translational modifications.

Example 19 Preparation of Tango-77 Proteins

Recombinant Tango-77 can be produced in a variety of expression systems.For example, the mature Tango-77 peptide can be expressed as arecombinant glutathione-S-transferase (GST) fusion protein in E. coliand the fusion protein can be isolated and characterized. Specifically,as described above, Tango-77 can be fused to GST and this fusion proteincan be expressed in E. coli strain PEB 199. Expression of theGST-Tango-77 fusion protein in PEB 199 can be induced with IPTG. Therecombinant fusion protein can be purified from crude bacterial lysatesof the induced PEB199 strain by affinity chromatography on glutathionebeads.

Example 20 Alternatively Spliced Forms of IL-1ra and Tango-77

Computer program Procrustes (Gelfand et al., 1996, Proc. Natl. Acad.Sci. USA, 93:9061-9066) is an alignment algorithm that predicts thepresence of alternatively spliced exons for a protein of interest in astretch of genomic DNA. Using the IL-1ra sequence, Proscustes was usedto search for the presence of additional sequences that might encode foralternatively spliced forms of IL-1ra in the two overlapping BAC genomicsequences. Potential sequences that encode variant exons for IL-1ra wereidentified. These predicted exons aligned well with the N-terminalregion of IL-1ra, but were not present in Tango-77. The results fromProcrustes predicts the existence of more spliced forms of IL-1ra.

Furthermore, Procrustes also predicted an additional sequence in BAC1(SEQ ID NO:86) and BAC2 (SEQ ID NO:87) that encodes an alternativelyspliced exon for Tango-77 (T77-procrustes). This predicted splicevariant form of Tango-77, T77-procrustes, was aligned with Tango-77 andwith IL-1ra and IL-1β.

PCR primers within this sequence can be used to generate a product thatcan be used to screen a panel of cDNA libraries using standardtechniques. Suitable cDNA libraries include libraries made fromTNFα-treated bronchoepithelium, TNFα-treated SSC cell line andanti-CD3-treated T cells. The resulting cDNA clone(s) can be isolatedfrom the library and sequenced to identify additional Tango-77 cDNAs.

Example 21 Isolation and Characterization of Human and Murine SPOILcDNAs

In this example, the isolation of the genes encoding human and murineSPOIL proteins (also referred to as “TANGO 080” proteins) are described.

Isolation of Murine SPOIL-I and SPOIL-II cDNAs

A murine SPOIL-I cDNA was identified by searching with a murine cDNAencoding an IL-1 signature region (Prosite™ Accession Number PDOC00226)against a copy of the GenBank nucleotide database using the BLASTN™program (BLASTN 1.3 MP: Altschul et al., J. Mol. Bio. 215:403, 1990). Aclone with 48% homology with the murine cDNA IL-1 signature region wasfound by this search. The sequence was analyzed against a non-redundantprotein database with the BLASTX™ program, which translates a nucleicacid sequence in all six frames and compares it against availableprotein databases (BLASTX 1.3 MP:Altschul et al., supra). This proteindatabase is a combination of the SwissProt, PIR, and NCBI GenPeptprotein databases. One clone was obtained from the IMAGE consortium, andfully sequenced. The additional sequencing of this clone extended theoriginal EST by 267 nucleotides at both the 5′ and 3′ ends. The cDNA forthis clone is approximately 746 nucleotides in length and has an openreading frame of 297 nucleotides that is predicted to encode a proteinof 98 amino acids.

The original first pass sequence of the clone showed homology to horseIL-1ra and murine IL-1ra using the BLASTX™ program. The nucleotidesequence and predicted amino acid sequences are shown in SEQ ID NO:89and SEQ ID NO:90, respectively. The murine SPOIL-I protein(corresponding to amino acids 1-98 of the predicted amino acid sequence,SEQ ID NO:90) shows 37.0% identity to the horse IL-1ra protein and 39.0%identity to the murine IL-1ra protein.

Alignment of murine SPOIL-I protein with murine IL-1α (SwissProt™Accession Number P01582) and murine IL-1β (SwissProt™ Accession NumberP10749) indicates the presence of an aspartic acid at amino acid residue91 of SEQ ID NO:90 and amino acid residue 74 of SEQ ID NO:93 whichcorresponds to an aspartic acid found at amino acid residue 266 ofmurine IL-1α and amino acid residue 261 of murine IL-1β. In addition,alignment of murine SPOIL-I with murine IL-1ra indicates that thisaspartic acid residue of SPOIL-I corresponds with a lysine at amino acidresidue 171 of murine IL-1ra (or amino acid residue 145 of mature murineIL-1ra) which has been shown to convert IL-1ra into an agonist bymutating this lysine residue to an aspartic acid residue. (Ju et al.(1991) Proc. Natl. Acad. Sci. USA 88:2658-2662).

This murine SPOIL-I protein contains an IL-1 signature domain(corresponding to amino acids 58-80 of the predicted amino acidsequence, SEQ ID NO:90 and amino acids 41-63 of SEQ ID NO:93) and asignal sequence (corresponding to amino acids 1-17 of the predictedamino acid sequence, SEQ ID NO:90) which is cleaved to form a matureSPOIL-I protein (corresponding to amino acids 1-81 of SEQ ID NO:93). Thepredicted molecular weight for the 98 amino acid SPOIL-I isapproximately 10.96 kDa and the predicted molecular weight for matureSPOIL-I (SEQ ID NO:93) is approximately 9.1 kDa.

A GenBank™ search using the murine SPOIL nucleotide sequence of SEQ IDNO:89 revealed a human EST (W78043) which was similar to a region of thenucleotide sequence of SEQ ID NO:89. As no reading frame can bedetermined from an EST (such as the EST identified in the above databasesearch) an amino acid sequence encoded by an EST can not be determined.

The entire cDNA of mouse SPOIL-I was used as a probe to screen a mouseesophagus library to search for alternate SPOIL transcripts. A secondform of mouse SPOIL was isolated and sequenced. This second form encodesa protein of 160 amino acid residues that lacks a signal peptide.Accordingly, this isoform, designated murine SPOIL-II is predicted to bean intracellular protein. Alignment of the 2 mouse SPOIL proteins (FIG.5B) shows that they are identical at the C-terminus but have differingN-termini. For example, murine SPOIL-I and SPOIL-II exhibit 100%identity when amino acid residues 29-98 of murine SPOIL-I are aligned toamino acid residues 91-160 of murine SPOIL-II . It is predicted that thetwo isoforms of murine SPOIL are splice variants of the murine SPOILgene.

A global alignment of murine SPOIL-I (SEQ ID NO:90) with murine SPOIL-II(SEQ ID NO:113) using the ALIGN program version 2.0 (global alignmentprogram, Myers and Miller, CABIOS, 1989) using a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 indicated thatthe proteins are 46.3% identical over the entire length of the sequences(FIG. 5B).

Isolation of Human SPOIL-I and SPOIL-II cDNAs

A cDNA library was constructed using mRNA isolated from near confluentmonolayers of human keratinocytes (Clonetics™) which had been stimulatedwith 50 ng/ml PMA, 1 μg/ml ionomycin, 10 ng/ml TNF, and 40 μg/mlcycloheximide for 4 hours. EST sequencing information was gathered tocreate a proprietary database of information describing the keratinocytecDNA clones. Three clones were identified by performing a TBLASTN searchof the proprietary EST database using the sequence of murine SPOIL-I asa query sequence (the three clones having a probability score of atleast 1.4e−48).

The nucleotide sequence and predicted amino acid sequences of humanSPOIL-I are shown in SEQ ID NO:101 and SEQ ID NO:102, respectively. Thenucleotide sequence and predicted amino acid sequences of human SPOIL-IIare shown in SEQ ID NO:104 and SEQ ID NO:105, respectively. A globalalignment of human SPOIL-I (SEQ ID NO:102) with human SPOIL-II (SEQ IDNO:105) using the ALIGN program version 2.0 (global alignment program,Myers and Miller, CABIOS, 1989) using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4 indicated that theproteins are 80.8% identical over the entire length of the sequences(FIG. 5A).

As was the case with the two murine isoforms of SPOIL, the two humanSPOIL isoforms exhibit exact identity at the C-terminus and are variantat their N-termini. Human SPOIL-II has an insertion of 40 amino acidresidues close to the N-terminus of the protein which are not present inhuman SPOIL-I. Like murine SPOIL-II, both human SPOIL isoforms lack asignal sequence, and accordingly, are predicted to be intracellularproteins. Human SPOIL-I and SPOIL-II may be splice variants of a commongene. An alignment of human SPOIL-I (SEQ ID NO:102) with murine SPOIL-I(SEQ ID NO:90) using the ALIGN program (parameters set as described forthe alignment of human SPOILS I and II) indicated that the proteins are26.3% identical over the entire length of the sequences, e.g., globalalignment. Moreover, using the same program and parameters, it wasdetermined that the nucleic acids which encode murine SPOIL-I (SEQ IDNO:89) and human SPOIL-II (SEQ ID NO:101) are 39.8% identical at thenucleotide level. An alignment of human SPOIL-II (SEQ ID NO:105) withmurine SPOIL-II (SEQ ID NO:113) using the ALIGN program (parameters setas described above) indicated that the proteins are 37.3% identical overthe entire length of the sequences, e.g., global alignment.

When locally aligned, the identity between the four SPOIL proteinsdescribed above is significant. TABLE VII sets forth the % identityamong SPOIL family members (when the C-terminal unique domains of eachfamily member are compared). Moreover, TABLE VII sets forth the %identity between each SPOIL C-terminal unique domain and murine IL-1ra.The alignment was performed using the Lipman-Pearson Algorithm (Lipmanet al. (1985) Science 227:1435-1441), with a K-tuple of 2, a Gap Penaltyof 4, and a Gap Weight Penalty of 12.

TABLE VII muSPOIL- muSPOIL- huSPOIL- huSPOIL- muIL- I II I II 1ramuSPOIL-I 100 muSPOIL-II 97.1 100 huSPOIL-I 52.2 53.6 100 huSPOIL-II52.2 53.6 100 100 muIL-ra 36.2 37.7 39.7 39.7 100

Alignment of the four SPOIL family members resulted in the generation ofat least two SPOIL consensus motifs, due to the highly conserved natureof specific amino acid residues among the family members. The SPOILconsensus motifs (“SPOIL signature motifs”) are set forth as SEQ IDNOs:110-111 (SEQ ID NO:110 corresponds to the short SPOIL signaturemotif and SEQ ID NO:111 corresponds to the long SPOIL consensus motif).Short and long SPOIL consensus motifs are found, for example, from aminoacid residues 26-69 and 26-93 of muSPOIL-I, from residues 88-131 and88-155 of muSPOIL-II, from residues 98-141 and 98-164 of huSPOIL-I, andfrom residues 137-180 and 137-203 of huSPOIL-II.

Further alignment of the intracellular SPOIL isoforms indicates that theproteins have at least 50% identity among the SPOIL unique domains ofthe proteins. TABLE VIII sets forth the % identity among SPOIL familymembers (when the SPOIL unique domains of each family member arecompared). The alignment was performed using the Lipman-PearsonAlgorithm (Lipmanet al. (1985) Science 227:1435-1441), with a K-tuple of2, a Gap Penalty of 4, and a Gap Weight Penalty of 12.

TABLE VIII muSPOIL-II huSPOIL-I huSPOIL-II muSPOIL-II 100 huSPOIL-I 50.3100 huSPOIL-II 50.3 100 100

Example 22 Distribution and Expression of SPOIL-I mRNA in Mouse andHuman Tissues In Situ Hybridization Analysis of Mouse Tissues

In situ analysis revealed the following expression patterns when tissuesections were hybridized with SPOIL-I probes. SPOIL-I mRNA was expressedalmost exclusively in the squamous epithelium of the esophagus in bothadult and embryonic mouse tissues. SPOIL-I mRNA was also expressed inthe epithelial lining of the mouth in adult mouse tissues and embryonicmouse tissues.

Moreover, in situ analysis of tissue samples for mice which had beenintravenously injected with 20 mg/kg of lippopolysaccharide (LPS)revealed that SPOIL-I expression was induced in the kidney.

Northern Blot Analysis of Human Tissues

Northern blot analysis of human tissues confirmed the pattern of SPOILexpression with SPOIL-I transcripts being detected in esophagus and,likely, trachea, among the tissues tested. In addition, SPOIL-I was alsopresent on human esophageal tumor samples and overexpressed inmoderately differentiated squamous cell carcinoma of the esophagus.

Expression of SPOIL in Human and Mouse Cell Lines

Human SPOIL-I expression was induced in keratinocytes (Clonetics) 2hours following induction with 50 ng/ml PMA, 1 ug/mL ionomycin, 10 ng/mlTNF and 40 ug/mL cyclohexamide. No expression was observed inunstimulated cultures.

Moreover, inducible expression of mouse SPOIL-I was observed in themonocytic cell line J774, 24 h after treatment with 0.1 μg/ml LPS.

Example 23 Expression of Recombinant SPOIL-I Protein in Bacterial Cells

SPOIL can be expressed as a recombinant glutathione-S-transferase (GST)fusion polypeptide in E. coli and the fusion polypeptide can be isolatedand characterized. Specifically, SPOIL is fused to GST and this fusionpolypeptide is expressed in E. coli, e.g., strain PEB199. As, forexample, the murine SPOIL-I protein is predicted to be approximately 9.1kDa and the GST is predicted to be approximately 26 kDa, the fusionpolypeptide is predicted to be approximately 35.1kDa in molecularweight. Expression of the GST-SPOIL-I fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB 199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 24 Expression of Recombinant SPOIL Proteins in COS Cells

To express the murine SPOIL-I gene, for example, in COS cells, thepcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used.This vector contains an SV40 origin of replication, an ampicillinresistance gene, an E. coli replication origin, a CMV promoter followedby a polylinker region, and an SV40 intron and polyadenylation site. ADNA fragment encoding the entire SPOIL-I protein and a HA tag (Wilson etal. (1984) Cell 37:767) fused in-frame to its 3′ end of the fragment iscloned into the polylinker region of the vector, thereby placing theexpression of the recombinant protein under the control of the CMVpromoter.

To construct the plasmid, the SPOIL-I DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the SPOIL-Icoding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag and the last 20nucleotides of the SPOIL-I coding sequence. The PCR amplified fragmentand the pcDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the SPOIL-I gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the SPOIL-I-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,1989. The expression of the SPOIL-I protein is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated proteins are then analyzed by SDS-PAGE.

Alternatively, DNA containing the SPOIL-I coding sequence is cloneddirectly into the polylinker of the pcDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of theSPOIL-I protein is detected by radiolabelling and immunoprecipitationusing a SPOIL-I specific monoclonal antibody

Example 25 Retroviral Delivery of SPOIL Proteins

Full length SPOIL-I genes were expressed in vivo by retroviral-mediatedinfection. In this example, the sequence for murine SPOIL-I (amino acids1-98) was amplified using the following primers;

Forward Primer (SEQ ID NO: 96): 5′AAAAAAGAAT TCGCCACCAT GTTCAGGATC TTA 3′ Reverse Primer (SEQ ID NO: 97):5′ TCCTCTGTCG ACTCACTTGT CGTCGTCGTC CTTGTAGTCA TGTACCACAA TCAT 3′

The reverse primer placed an epitope tag (Flag sequence) on the 3′ endof the protein. Amplified products were then subcloned into theretroviral vector MSCVneo (Hawley et al. (1994) Gene Therapy 1:136-138),and sequence verified. Bone marrow from 5-fluorouracil treated miceinfected with the retrovirus was then transplanted into irradiated mouserecipients and the pathology was reviewed after 5 weeks.

The spleen and bones of the mouse recipients were taken 5 weeks aftertransplantation. Disassociated spleen cells, which are a source ofosteoclast progenitors, from the SPOIL-I infected mice were plated ontop of ST2 bone marrow stromal line in the presence of 1, 25dihdroxyvitamin D3 as described by Lacey et al. (1995) Endocrinology136:2367-2376 and Udagawa et al. (1989) Endocrinology 125:1805-1813. Inaddition, spleen cells from control mice transplanted with marrowinfected with retrovirus without the inserted SPOIL-I gene, were plated.After nine days of culture, the number of osteoclasts was determined bystaining for tartrate resistant acid phosphatase (TRAP).

The results of these experiments demonstrated that the number of TRAPpositive osteoclasts was dramatically decreased in cultures with theSPOIL-I infected spleen cells as compared to the control cells.Histologically, the bones of mice recipients transplanted with SPOIL-Iinfected marrow, also appeared to be thicker than the bones of thecorresponding control mice. Generally, there was less trabecular bone atthe growth plate. The trabecular bone was compressed and thickened withmore osteoloid formation and more osteoblasts present.

Example 26 Identification And Characterization of NEOKINE-1 cDNAs

In this example, the identification and characterization of the genesencoding human, murine, rat and macaque NEOKINE-1 (also referred to as“ANTIKINE-1”, “TANGO 112”, or T112) is described.

Isolation of the Murine and Human NEOKINE-1 cDNAs

The invention is based, at least in part, on the discovery of the murineand human genes encoding a novel protein, referred to herein asNEOKINE-1. In order to identify potentially novel chemokines, using anautomated procedure, the human amino acid sequences of the chemokines,interleukin-8, gamma-IP10, Sis-Delta, fractalkine, and SDF-1 were usedto search proprietary databases and the dbEST databases using TBLASTN(Wash U. version, 2.0, BLOSUM62 search matrix). Sequences exhibiting 90%or greater identity to any protein present in Genpept, SwissProt, or PIRwere marked as examples of these proteins and removed. This analysisidentified a mouse EST (accession number AA013634) potentially encodinga chemokine. As the encoded protein was quite divergent from all otherchemokine family members, and the open reading frame constituted a smallpercentage of the total cDNA length (see below), to establish whetherthis transcript encoded a novel chemokine family member, the nucleotidesequence of the entire cDNA was determined. To do this, first,additional ESTs from the mouse gene were retrieved from public databasesby similarity searches. A total of 33 murine sequences were thusretrieved. Second, these sequences were used to create an extensivelength of continuous sequence (contig). The 33 murine ESTs were alignedand edited at discrepant bases into a single contig of 1420 bp. The 5′end of the contig appeared to be missing part of the open reading frame.To extend this sequence, the sizes of the inserts of several of themurine cDNA clones from which the ESTs were derived was determined andone with the largest insert (about 1.45 kb) was subsequentlyre-sequenced in its entirety. This extended the sequence by 5 by to 1425bp and corrected some remaining discrepancies.

72 human ESTs from a presumptive human orthologue of the above-describedmouse gene were identified. However, these ESTs, after a similarassembly and editing, aligned into two non-overlapping contigs of 364and 1101 bp. Comparison with the murine sequence suggested that the364-bp contig derived from the 5′ part of the human gene, while the1101-bp contig derived from the 3′ part of the human gene and includedthe poly(A) tail. The distribution of 5′ EST reads implied that therewas no cDNA clone which contained both halves of the gene. At the 3′ endof the 5′ contig, there was a naturally occurring run of adenosinenucleotides (see below), to which the oligo-d(T) primer used in reversetranscription would have annealed (i.e., this primer would have annealedat two sites: the actual poly (A) tail and this internal oligo (A)stretch). This ectopic primer would have blocked reverse transcriptionin the human sequence at about 1.1 kb from the 3′ end, but also wouldhave allowed a second set of cDNA molecules to be synthesized whichcovered the 5′ end of the gene. The two human EST contigs that can bederived from these 72 ESTs therefore would appear on inspection and inthe absence of further information, to derive from two genes instead ofone. That this is the case is shown by the fact that automated assemblyof ESTs produced distinct “UniGene” numbers for the two contigs(Hs.21210 for the 5′ contig and Hs.24395 for the 3′ contig).

To establish that the two human contigs derived from the sametranscript, two primers lacking oligo d(T) were designed from the twoassembled human contigs such that they would amplify an ˜300 bp fragmentspanning the contigs. Using first-strand cDNA prepared from humanplacental poly (A)+ RNA, a unique ˜300 bp fragment aws amplified bystandard techniques. This cDNA was subsequently cloned and sequenced.The sequence did span the two contigs and provided the missing sequencebetween them. The primers were h112/227f (CCAAGCGCTTCATCAAGTGG) (SEQ IDNO:125) and h112/526r (GCAGCCTGTGATGAAGTCTGG) (SEQ ID NO:126).

The 5′ end of the contig, which included part of the open reading frame,was missing from the assembled single contig. To obtain the completetranscript sequence at the 5′ end for the human gene, a cDNA clone(rthp112) extending from the 5′ end of the human transcript to beyondthe end of the open reading frame was generated by the RACE procedure.The gene-specific primer used was t112racel (CAGCCTATTCTTCGTAGACCCTGC)(SEQ ID NO:127). The 5′-most 35 bp of this clone formed a palindrome andthus appeared to be a cloning artifact, as is well known in the art, andwas removed from the final sequence. The remaining sequence extended thehuman cDNA sequence by 108 bp to 1564 bp and corrected some remainingdiscrepancies. Clone rthp112, comprising the entire coding region ofhuman NEOKINE-1 has been deposited with the ATTC and has Accession No.98751.

The nucleotide sequence encoding the human NEOKINE-1 protein is setforth as SEQ ID NO:115. The full length protein encoded by this nucleicacid is comprised of about 99 amino acids and has the amino acidsequence set forth as SEQ ID NO:116. The coding portion (open readingframe) of SEQ ID NO:115 is set forth as SEQ ID NO:117.

To identify a murine cDNA clone containing a near full-length insert,the sizes of the inserts of several of the murine cDNA clones from whichthe ESTs were derived was determined and one with the largest insert(about 1.45 kb) was subsequently re-sequenced in its entirety. Thisextended the sequence by 5 bp to 1425 bp and corrected some remainingdiscrepancies.

The nucleotide sequence encoding the murine NEOKINE-1 protein is setforth as SEQ ID NO:118. The full length protein encoded by this nucleicacid is comprised of about 92 amino acids and has the amino acidsequence set forth as SEQ ID NO:119. The coding portion (open readingframe) of SEQ ID NO:118 is set forth as SEQ ID NO:120.

Analysis of Murine and Human NEOKINE-1

Examination of the assembled and corrected cDNA sequences depicted inSEQ ID NO:115 and SEQ ID NO:118 showed that they likely encodedhighly-conserved proteins, human and mouse NEOKINE-1. Based on thepresence of 4 cysteine residues, which presumably form 2 disulfidebonds, a predicted signal sequence, a predicted mature peptide mass ofabout 10,000 daltons, and a characteristic spacing of one residuebetween the first two cysteines, it was judged that the encoded proteinwas a novel member of the alpha chemokine family, and a member of thesubfamily that lacked the glutamine-leucine-arginine sequence before thefirst cysteine. However, three atypical features were also present butconserved between species. These were, first, the presence of an extra 5residues between the second pair of cysteines; second, the fewestresidues before the predicted amino terminus of the mature protein andthe first cysteine of any naturally-occurring chemokine; and third, ageneral dissimilarity to all other chemokines in the region between thesecond and third cysteines.

A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of thenucleotide sequence of human NEOKINE-1 has revealed that NEOKINE-1 issignificantly similar to a human STS (TIGR-A002114, Accession No.G26440) which was sequenced as part of the WI/MIT human gene mappingproject and derived from a TIGR-assembled contig that lacks any of theopen reading frame of human NEOKINE-1. (The TIGR-assembled contig failedto reveal the true ORF of human NEOKINE-1, most likely to the existenceof a significant number of potential ORFs which fortuitously exist inthe long 3′ UTR of the human NEOKINE cDNA, but do not, in fact encodethe human NEOKINE-1 protein.) The gene is located to human 5q31.1 nearthe marker D5S396, distinct from the chemokine cluster on chromosome 4q.Plausible human disease genes that map to this region include ahereditary eosinophilia (EOS) and a hereditary high serum IgE associatedwith hypersuppression of inflammation in the skin (IGES).

The clones of both the human and murine EST sequences used in theassembly of the human and murine contigs derive predominantly fromprenatal tissues. In particular, 6 human ESTs derive from clonesisolated from neonatal female placenta, 4 human ESTs derive from clonesisolated from 8-9-week placenta, 4 human ESTs derive from clonesisolated from fetal heart, 4 human ESTs derive from clones isolated from20-week male liver and spleen, 4 human ESTs derive from clones isolatedfrom breast tumor, 4 human ESTs derive from clones isolated from colontumor, 3 human ESTs derive from clones isolated from adult breast, 2human ESTs derive from clones isolated from pregnant uterus, 2 humanESTs derive from clones isolated from endometrial tumor, 1 human ESTderives from a clone isolated from fetal brain, 1 human EST derives froma clone isolated from alveolar rhabdomyosarcoma, 1 human EST derivesfrom a clone isolated from ovary tumor, 1 human EST derives from a cloneisolated from 8-9-week total fetus, 1 human EST derives from a cloneisolated from TIGR placenta II, 1 human EST derives from a cloneisolated from corneal stroma, 1 human EST derives from a clone isolatedfrom 3 m muscular atrophy, 1 human EST derives from a clone isolatedfrom thyroid tumor, 1 human EST derives from a clone isolated from a6-week embryo, and 1 human EST derives from a clone isolated from a12-week embryo. murine ESTs derive from clones isolated from 13.5+14.5dwhole embryo, 5 murine ESTs derive from clones isolated from 19.5dembryo, 4 murine ESTs derive from clones isolated from 8.5d embryo, 3murine ESTs derive from clones isolated from 12.5d embryo, 3 murine ESTsderive from clones isolated from 7d kidney. 2 murine ESTs derive fromclones isolated from 13.5+14.5d placenta, 1 murine EST derives from aclone isolated from 6.5/8.5d embryo, 1 murine EST derives from a cloneisolated from liver, 1 murine EST derives from a clone isolated from4-week male thymus, 1 murine EST derives from a clone isolated fromdiaphragm, and 1 murine, EST derives from a clone isolated from 11-weekskin.

Tissue Distribution of NEOKINE-1 mRNA

This Example describes the tissue distribution of NEOKINE mRNA, asdetermined by Northern blot and in situ hybridization.

Northern blot hybridizations with the various RNA samples were performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. In each sample, the probe hybridized to a single RNAof about 1.9 kb. The results of hybridization of the probe to variousmRNA samples are described below.

Expression in mouse embryos was examined using a developmental mouseNorthern (Clontech) and in situ hybridization. This blot had poly(A)+RNA isolated from whole embryos of age 7, 11, 15 and 17 days. Theanalysis using the mouse gene as a probe revealed intense expression ofa unique 1.9 kb transcript in the 7 d sample, but this may be due tocontaminating RNA from the placenta. In the remaining samples,expression was low at 11d, highest on day 15, and then dropped again onday 17.

Expression in diverse human tissue was examined using tissue-specificNorthern blots (Clontech). These blots had poly(A)+ RNA isolated fromvarious disease-free human organs. The analysis using a PCR fragment ofthe human gene as a probe revealed strongest expression of a unique 1.9kb transcript in the kidney and small intestine, followed by strongexpression in the spleen, uterus and colon, and lower expression inthymus, prostate, stomach, thyroid, spinal cord, lymph node, trachea,adrenal gland, bone marrow, heart, brain placenta, liver, smooth muscleand pancreas. There was little/no expression in lung, peripheral bloodleukocytes and testis. The size of the poly(A)+transcript in both humanand mouse (1.9 kb) is consistent with the length of the full-lengthcDNAs (1.56 kb) which lack the poly(A) tails.

In situ hybridization of a murine antisense probe to various embryonic,post-natal and adult tissues was performed as follows. 8 μm sagittalsections of fresh frozen embryonic day 14.5, 16.5 and postnatal day 1.5B6 mice, as well as 8 μm sections of various adult B6 mouse tissues(listed below) were used for hybridization. Sections were postfixed with4% formaldehyde in DEPC treated 1× phosphate-buffered saline at roomtemperature for 10 min before being rinsed twice in DEPC 1×phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH8.0). Following incubation in 0.25% acetic anhydride-0.1 Mtriethanolamine-HCl for 10 min, sections were rinsed in DEPC 2×SSC(1×SSC is 0.15M NaCl plus 0.015M sodium citrate). Tissue was dehydratedthrough a series of ethanol washes, incubated in 100% chloroform for 5min, and then rinsed in 100% ethanol for 1 min and 95% ethanol for 1 minand allowed to air dry.

The hybridization was performed using a ³⁵S-radiolabeled cRNA(antisense) probe from the following DNA sequence,

(SEQ ID NO: 128) GTCCAAGTGTAAGTGTTCCCGGAAGGGGCCCAAGATCCGCTACAGCGACGTGAAGAAGCTGGAAATGAAGCCAAAGTACCCACACTGCGAGGAGAAGATGGTTATCGTCACCACCAAGAGCATGTCCAGGTACCGGGGCCAGGAGCACTGCCTGCACCCTAAGCTGCAGAGCACCAAACGCTTCATCAAGTGGTACAATGCCTGGAACGAGAAGCGCAGGGTCTACGAAGAATAGGGTGGACGATCATGGAAAGAAAAACTCCAGGCCAGTTGAGAGACTTCAGCAGAGGACTTTGCAGATTAAAATAAAAGCCCTTTCTTTCTCACAAGCATAAGACAAATTATATATTGCTATGAAGCTCTTCTTACCAGGGTCAGTTTTTACATTTTATAGCTGTGTGTGAAAGGCTTCCAGATGTGAGATCCAGCTCGCCTGCGCACCAGACTTCATTACAAGTGGCTTTTTGCTGGGCGGTTG.

A sense RNA probe made from the same DNA sequence was used to determinespecificity of the antisense probe. Tissues were incubated with probes(approximately 5×10⁷ cpm/ml) in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1×Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hat 55° C. After hybridization, slides were washed with 2×SSC. Sectionswere then sequentially incubated at 37° C. in TNE (a solution containing10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 min, in TNEwith 10 μg of RNase A per ml for 30 min, and finally in TNE for 10 min.Slides were then rinsed with 2×SSC at room temp, washed with 2×SSC at50° C. for 1 h, washed with 0.2×SSC at 55° C. for 1 h, and 0.2×SSC at60° C. for 1 h. Sections were then dehydrated rapidly through serialethanol-0.3 M sodium acetate concentrations before being air dried andexposed to Kodak Biomax MR scientific imaging film for 4 days at roomtemperature.

Following a 4 day film exposure, NEOKINE mRNA was detectable in thefollowing tissues (Note: Tissues incubated with sense probe showed nosignal in any tissues):

Adult Mouse:

Brain—multifocal signal in the cortexPurkinje cell layer of the cerebellumareas of the hippocampus and forebraina small discrete region in the ventral portion of the hindbrainlow level ubiquitous signal in most other regionseye—multifocal signal seen in harderian glandanterior surface of lens/cornearetinadescending colon—focal signaltransverse/ascending colon—multifocal signalsmall Intestine—villikidney—cortical regionadrenal gland—medulla region and capsuleheart—multifocal signalskeletal muscle—multifocal signallung—signal outlining the large airwaysthymus—low signalbladder—high signal from the transitional epitheliumplacenta—signal seen in the outer membrane/cell layerThe following tissues were tested but no signal was detected in:spleenliverPostnatal Day 1.5 mouse:brain—specific regions, most notably the cortexno ubiquitous expression as seen in adultnasal turbinatesdeveloping upper and lower teethtracheauterussternebral cartilagekidney—medulla and outermost cortex in a multifocal patternskin and hair follicles—very strong signalintestinenote: no signal observed in lungEmbryonic Day 16.5 mouse:brain—specific regions, most notably the cortexno ubiquitous expression as seen in adultspinal cord—low signalesophaguslung—signal from large airwaysadrenal gland—cortical region (note: opposite of adult)kidney—medulla and outermost cortex in a multifocal patternskin—very strong signalintestineEmbryonic Day 14.5 mouse:brain—discrete regions, most notable a region in the hindbrainlung—signal from large airwaysskin—very strong signal especially from whisker pads and tip of nose andtailumbilical cordintestine

These results reveal a striking distribution of NEOKINE mRNA in variousnon-lymphoid organs. The fact that many normal tissues express abundantNEOKINE transcript suggests that these tissues make significantquantities of NEOKINE protein and that therefore the pro-inflammatory orchemoattractive activities of NEOKINE are very low or non-existent.Indeed, NEOKINE is expressed most significantly in highly immunoreactivetissues that are involved in barrier functions. This observation isconsistent with a proposed role of NEOKINE as a suppressor ofinflammation. Furthermore, the predicted amino terminus of matureNEOKINE lies just two residues from the first cysteine. In an analogoussituation, an artificially truncated form of human IL-8 with only oneresidue before the first cysteine instead of the normal 6 residuesconverts the protein from an agonist to an antagonist of its cognatereceptor. These observations are further consistent with the proposedanti-inflammatory activity being mediated by antagonizing the action ofother pro-inflammatory chemokines. The general divergence of NEOKINEfrom other alpha chemokines while being highly conserved itself, and thepresence of the extra 5 residues between the second pair of cysteinescould also be consistent with a broad antagonist function on a diverseset of chemokine receptors. The strong expression in the skin, thekidney, the bronchii and the brain indicates that NEOKINES have utilityin treating inflammation of these organs, such as occurs in acute renalfailure, transplantation, allergy and infection. Furthermore, since thehuman AIDS virus HIV uses some chemokine receptors as co-receptors forinfection, NEOKINES may also have utility in slowing or blockinginfection by HIV.

Example 27 Isolation And Characterization of Rat and Macaque NEOKINE-1cDNAs

During routine tests for sequences similar to mouse or human NEOKINEcDNA, one EST deriving from a rat brain cDNA and three ESTs derivingfrom macaque brain cDNAs were identified in a database of proprietarysequences. The database derives, at least in part, from sequencing ofvarious mammalian clones generated from cDNA libraries created accordingto routine procedures. The cDNAs originated from rat brain and macaquebrain libraries, respectively.

The nucleotide sequence encoding the at least 78 amino acid residues ofthe rat NEOKINE-1 protein (corresponding to the predicted matureprotein) is set forth as SEQ ID NO:121. The amino acid sequence formature rat NEOKINE-1 is set forth as SEQ ID NO:122. The coding portion(open reading frame) of SEQ ID NO:121 is set forth as SEQ ID NO:123. Thenucleotide sequence and predicted amino acid sequence of macaqueNEOKINE-1 are set forth as SEQ ID NO:124 and SEQ ID NO:135,respectively.

Examination of the cDNA sequence depicted in SEQ ID NO:121 shows thatrat NEOKINE-1 comprises four cysteine residues which are conserved amongall NEOKINE-1 family members identified thus far. Structural analysis ofthese proteins indicates that these cysteines are capable of forming 2disulfide bridges. FIG. 8 depicts the alignment between the fourNEOKINE-1 amino acid sequences identified according to these Examples.

Example 28 Secretion of NEOKINE Chemokines

Expression constructs for RGSHis6 epitope-tagged (C-terminus) humanNEOKINE were transfected into 293T cells using lipofectamine (GIBCO/BRL)according to the manufacturers instructions. After culturing inappropriate medium for 48-72 hours, conditioned medium was harvested,spun, filtered, and passed over nickel metal chelating column (Qiagen).After washing, bound material was eluted 200 mM imidazole buffer andfractions collected. Peak fractions were analyzed by SDS-PAGE andwestern blot using anti His6 antibodies (Quiagen). Purified NEOKINEprotein bound to PVDF membrane after SDS-PAGE and electroblotting wassequenced for N-terminal amino acid analysis using Edman-based chemistryprotein sequencing. The amino acid residues were analyzed by HPLC anddetermined by separation and peak height as compared to standards.

The N-terminal sequence of band NEOKINE was found to be SKCKCSRKGP whichcorresponds exactly to the predicted signal peptide cleavage site(between Gly22 and Ser23). Because the same band is identified byanti-His6 antibodies, which recognize the C-terminal epitope tag, theband was identified as the full length, mature NEOKINE protein.

Example 29 Binding of NEOKINE to the NEOKINE Receptor (e.g., RDC1)

T112 (NEOKINE) was radioiodinated with lactoperoxidase according tostandard protocols. RDC-1 in vector Pcdna3.1 was transiently transfectedinto 293 cells using calcium phosphate precipitation methodology. 72hours after transfection, cells were harvested and binding assaysperformed under standard binding conditions for chemokines (e.g., lowsalt binding, high salt wash, binding at 24° C., 1 hour). Cells werethen pelleted, washed, and radioactivity was counted. Binding wasdemonstrated and was determined to be high affinity by competition withunlabelled T112. The cpm bound in cell pellets in indicated below.

¹²⁵I-T112 0.1 nm 37613.0 cpm 27014.0 cpm T112 3.0 nm  8343.0 cpm  9229.0cpm

Example 30 Isolation and Characterization of Human T129 cDNAs

Human mesangial cells (Clonetics Corporation; San Diego, Calif.) wereexpanded in culture with Mesangial Cell Growth Media (Clonetics)according to the recommendations of the supplier. When the cells reached80-90% confluence, they were stimulated with tumor necrosis factor (TNF;10 ng/ml) and cycloheximide (CHI; 40 micrograms/ml) for 4 hours. TotalRNA was isolated using the RNeasy Midi Kit (Qiagen; Chatsworth, Calif.),and the poly A+ fraction was further purified using Oligotex beads(Qiagen).

Three micrograms of poly A+ RNA were used to synthesize a cDNA libraryusing the Superscript cDNA Synthesis kit (Gibco BRL; Gaithersburg, Md.).Complementary DNA was directionally cloned into the expression plasmidpMET7 using the SalI and NotI sites in the polylinker to construct aplasmid library. Transformants were picked and grown up for single-passsequencing.

One clone, jthKb042d12, showed limited homology to OX40 (Latza et al.(1994) Eur. J. Immunol. 24:677), a member of the TNF receptorsuperfamily, and was sequenced further. Complete sequencing of the clonerevealed an approximately 2.5 kb cDNA insert with a 1290 base pair openreading frame predicted to encode a novel 430 amino transmembraneprotein.

Example 31 Distribution of T129 mRNA in Human Tissues

The expression of T129 was analyzed using Northern blot hybridization. A567 bp portion of T129 cDNA encoding the amino terminus of T129 proteinwas generated by PCR. The DNA was radioactively labeled with ³²P-dCTPusing the Prime-It kit (Stratagene; La Jolla, Calif.) according to theinstructions of the supplier. Filters containing human mRNA (MTNI andMTNII: Clontech; Palo Alto, Calif.) were probed in ExpressHybhybridization solution (Clontech) and washed at high stringencyaccording to manufacturer's recommendations.

These studies revealed that T129 is expressed as an approximately 3.0kilobase transcript at moderate levels in peripheral blood leukocytes,spleen, and skeletal muscle. Lower levels of transcript were seen inheart, brain and placenta. In addition, a hybridization signal was seenin peripheral blood leukocytes at >15 kb.

Example 32 Characterization of T129 Proteins

In this example, the predicted amino acid sequence of human T129 proteinwas compared to amino acid sequences of known proteins and variousmotifs were identified. In addition, the molecular weight of the humanT129 proteins was predicted.

The human T129 cDNA isolated as described above (SEQ ID NO:137) encodesa 430 amino acid protein (SEQ ID NO:138). The signal peptide predictionprogram SIGNALP Optimized Tool (Nielsen et al. (1997) ProteinEngineering 10:1-6) predicted that T129 includes a 22 amino acid signalpeptide (amino acid 1 to about amino acid 22 of SEQ ID NO:137) precedingthe 408 mature protein (about amino acid 23 to amino acid 430; SEQ IDNO:140). T129 also include one predicted transmembrane domain (aminoacids 163-186 of SEQ ID NO:138). A hydropathy plot of T129 is presentedin FIG. 9. This plot shows the two predicted TM domains as well as aextracellular region (labelled “OUT”; amino acids 31 to 162 of SEQ IDNO:138) and a cytoplasmic region (labelled “IN”; amino acids 187 to 430of SEQ ID NO:138) as well as the location of cysteines (“cys”; shortvertical lines just below plot) and the TNFR/NGFR cysteine-rich domainindicated by its PFAM identifier (PF0020; bar just above plot). Forgeneral information regarding PFAM identifiers refer to Sonnhammer etal. (1997) Protein 28:405-420.

T129 has a region (amino acids 51-90; SEQ ID NO:142) of homology to aTNFR/NGFR cysteine-rich domain consensus derived from a hidden Markovmodel (SEQ ID NO:141). The TNFR/NGFR cysteine-rich domain of T129 doesnot include all the conserved cysteines usually present in such domains(4 of 6). Moreover, unlike other members of the TNF superfamily, T129includes only one such domain; most TNF family members include two tofour such cysteine rich domains.

Mature T129 has a predicted MW of 43.5 kDa (46 kDa for immature T129),not including post-translational modifications.

Example 33 Preparation of T129 Proteins

Recombinant T129 can be produced in a variety of expression systems. Forexample, the mature T129 peptide can be expressed as a recombinantglutathione-S-transferase (GST) fusion protein in E. coli and the fusionprotein can be isolated and characterized. Specifically, as describedabove, T129 can be fused to GST and this fusion protein can be expressedin E. coli strain PEB199. Expression of the GST-T129 fusion protein inPEB 199 can be induced with IPTG. The recombinant fusion protein can bepurified from crude bacterial lysates of the induced PEB 199 strain byaffinity chromatography on glutathione beads.

Deposit of Microorganisms

A nucleic acid encoding a full-length human Delta protein is containedin a plasmid which was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 (ATCC®)on Mar. 5, 1997 and has been assigned ATCC® accession number 98348.

A clone containing the cDNA molecule encoding human A259, (clone Human12) was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va., 20110-2209, on Apr. 2, 1999 asAccession Number 207190, as a single deposit.

A clone containing the cDNA molecule encoding mouse A259, (clone Mouse12) was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va., 20110-2209, on Apr. 2, 1999 asAccession Number 207191, as a single deposit.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein.

Such equivalents are intended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1-12. (canceled)
 13. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 80% identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3; b) a nucleic acid molecule comprising a fragment of at least 500 nucleotides of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 100 contiguous amino acids of SEQ ID NO:2; and e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, or a complement thereof, under stringent conditions.
 14. The isolated nucleic acid molecule of claim 13, further comprising a fragment of at least 1000 nucleotides of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
 15. The isolated nucleic acid molecule of claim 13, further comprising a fragment of at least 1500 nucleotides of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
 16. The isolated nucleic acid molecule of claim 13, further comprising a fragment of at least 2000 nucleotides of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
 17. The isolated nucleic acid molecule of claim 13, which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 100 contiguous amino acids of SEQ ID NO:2.
 18. The isolated nucleic acid molecule of claim 13, which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 200 contiguous amino acids of SEQ ID NO:2.
 19. The isolated nucleic acid molecule of claim 13, which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
 20. The nucleic acid molecule of claim 13 further comprising vector nucleic acid sequences.
 21. The nucleic acid molecule of claim 13 further comprising nucleic acid sequences encoding a heterologous polypeptide.
 22. A host cell which contains the nucleic acid molecule of claim
 13. 23. The host cell of claim 22 which is a mammalian host cell.
 24. A non-human mammalian host cell containing the nucleic acid molecule of claim
 13. 25. An isolated polypeptide selected from the group consisting of: a) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 80% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or SEQ ID NO:3; and c) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 100 contiguous amino acids of SEQ ID NO:2.
 26. The isolated polypeptide of claim 25, comprising a fragment which comprises at least 100 contiguous amino acids of SEQ ID NO:2.
 27. The isolated polypeptide of claim 25, comprising a fragment which comprises at least 200 contiguous amino acids of SEQ ID NO:2.
 28. The isolated polypeptide of claim 25, comprising a fragment which comprises at least 500 contiguous amino acids of SEQ ID NO:2.
 29. The isolated polypeptide of claim 25, comprising a fragment which is at least 90% homologous to the amino acid sequence of SEQ ID NO:2.
 30. The isolated polypeptide of claim 25, comprising a fragment which is at least 95% homologous to the amino acid sequence of SEQ ID NO:2.
 31. The isolated polypeptide of claim 25, comprising the amino acid sequence of SEQ ID NO:2.
 32. The isolated polypeptide of claim 25 further comprising heterologous amino acid sequences.
 33. An antibody which selectively binds to a polypeptide of claim
 25. 34. The antibody of claim 33, which is a monoclonal antibody.
 35. The antibody of claim 34, comprising an immunologically active portion selected from the group consisting of: a) an scFV fragment; b) a dcFV fragment; c) an Fab fragment; and d) an F(ab′).sub.2 fragment.
 36. The antibody of claim 34, wherein the antibody is selected from the group consisting of: a) a chimeric antibody; b) a humanized antibody; c) a human antibody; d) a non-human antibody; and e) a single chain antibody.
 37. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 100 contiguous amino acids of SEQ ID NO:2; c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC as Accession Number ______, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, or a complement thereof under stringent conditions; comprising culturing the host cell of claim 22 under conditions in which the nucleic acid molecule is expressed.
 38. A method for detecting the presence of a polypeptide of claim 25 in a sample, comprising: contacting the sample with a compound which selectively binds to a polypeptide of claim 25; and determining whether the compound binds to the polypeptide in the sample.
 39. The method of claim 38, wherein the compound which binds to the polypeptide is an antibody.
 40. A kit comprising a compound which selectively binds to a polypeptide of claim 25 and instructions for use.
 41. A method for detecting the presence of a nucleic acid molecule of claim 13 in a sample, comprising the steps of: contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
 42. The method of claim 41, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
 43. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 13 and instructions for use.
 44. A method for identifying a compound which binds to a polypeptide of claim 25 comprising the steps of: contacting a polypeptide, or a cell expressing a polypeptide of claim 25 with a test compound; and determining whether the polypeptide binds to the test compound.
 45. The method of claim 44, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; and c) detection of binding using an assay for Delta3-mediated signal transduction.
 46. A method for modulating the activity of a polypeptide of claim 25 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 25 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
 47. A method for identifying a compound which modulates the activity of a polypeptide of claim 25, comprising: contacting a polypeptide of claim 25 with a test compound; and determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
 48. An isolated antibody, or portion thereof, that specifically binds to a polypeptide selected from the group consisting of: a) the polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) the polypeptide encoded by the nucleic acid molecule of SEQ ID NO: 1 or 3; and c) the polypeptide encoded by the nucleotide sequence contained in the plasmid deposited with the ATCC as Accession number
 98348. 49. The antibody, or portion thereof, of claim 48, wherein said antibody specifically binds to the polypeptide comprising the amino acid sequence of SEQ ID NO:2.
 50. The antibody, or portion thereof, of claim 48, wherein said antibody specifically binds to the polypeptide encoded by the nucleic acid molecule of SEQ ID NO: 1 or
 3. 51. The antibody, or portion thereof, of claim 48, wherein said antibody specifically binds to the polypeptide encoded by the nucleotide sequence contained in the plasmid deposited with the ATCC as Accession number
 98348. 52. The antibody, or portion thereof, of claim 48, wherein said antibody is selected from the group consisting of: i) a monoclonal antibody; ii) a polyclonal antibody; iii) a bispecific antibody; and iii) a chimeric antibody.
 53. The antibody, or portion thereof, of claim 48, wherein said portion is a F(ab) fragment or a F(ab′).sub.2 fragment.
 54. The antibody, or portion thereof, of claim 48, wherein said antibody binds to amino acid residues 18-529 of SEQ ID NO:2.
 55. The antibody, or portion thereof, of claim 48, wherein said antibody is detectably labeled.
 56. The antibody, or portion thereof, of claim 55, wherein the detectable label is selected from the group consisting of: a) an enzyme; b) a fluorescent material; c) a luminescent material; d) a bioluminescent material; and e) a radioactive material. 